Factors from damaged sperm affect its DNA integrity and its ability to promote embryo implantation in mice

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P < .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.     

Does seminal plasma PSP-I/PSP-II spermadhesin modulate the ability of boar spermatozoa to penetrate homologous oocytes in vitro?

Low concentration (0.15 mg per million of spermatozoa) of seminal plasma-derived PSP-I/PSP-II spermadhesin heterodimer is able to preserve the viability of highly extended boar spermatozoa. Whether spermatozoa also keep their fertilizing capacity is not yet known. The present study evaluated the effect of exposing freshly extended and frozen-thawed boar spermatozoa (10 million/mL) to PSP-I/PSP-II (1.5 mg/mL) for 30 or 120 minutes on sperm characteristics and the outcome of in vitro penetration of immature (IM) and in vitro matured (IVM) homologous oocytes, aiming to identify this spermadhesin as a suitable modulator for sperm-handling protocols. Although exposure to the heterodimer improved sperm viability and motility without increasing the levels of sperm acrosome exocytosis in both freshly extended and frozen-thawed spermatozoa, this pretreatment did not affect sperm penetration rates or sperm numbers per oocyte when pretreated fresh spermatozoa were coincubated with IM or IVM oocytes compared with controls. When cryopreserved spermatozoa were tested, however, on IVM oocytes, already a 30-minute preincubation exposure to PSP-I/PSP-II showed a significant blocking effect on penetration rate (from 90% to 32%, P < .05) and on mean sperm numbers per oocyte (2.9 to 1.6, P < .05). To disclose the nature of this paradox, frozen-thawed spermatozoa were cleansed (by centrifugation in saline bovine serum albumin or through Percoll density gradient separation) and the procedure repeated. Oocyte penetration (but not number of spermatozoa per oocyte) increased (P < .05) when spermatozoa were cleansed with Percoll compared with either washed or unwashed controls (53% vs 13% vs 31%, respectively). In addition, the percentages of polyspermic oocytes remained lower than control (38.5% vs 68.7%, respectively; P < .05). In conclusion, the results confirm that exposure of fresh or frozen-thawed boar spermatozoa to a low dose of seminal PSP-I/PSP-II spermadhesin preserves sperm viability and motility in vitro. Although there was no obvious influence of the heterodimer on the capability of freshly extended boar spermatozoa to penetrate homologous oocytes (either IM or IVM), PSP-I/PSP-II exerted a deleterious effect when frozen-thawed spermatozoa were used to penetrate IVM oocytes. Such an effect of cryopreservation seems to a certain extent reversible, since cleansing of the sperm surface decreased, at least partially, this blocking effect, increasing both penetration and the monospermic rates.     

The scheduled ovarian hyperstimulation method makes it easy to perform ICSI with fresh testicular sperm (ICSI/TESE)

The authors evaluated whether scheduled ovarian stimulation makes it easy to perform ICSI with fresh testicular sperm. Scheduled ovarian hyperstimulation was applied for testicular sperm extraction and ICSI with fresh testicular spermatozoa. Fifteen cycles in 10 couples were included in the present study; all couples were azoospermic, 5 were obstructive, and the remaining 5 were nonobstructive. No cycles were canceled, and all oocyte retrievals were performed on the scheduled day. Testicular sperm were obtained in 14 treatment cycles (93%). The mean numbers of retrieved and injected oocytes were 9.4 and 6.4, respectively. The fertilization and cleavage rates were 47 and 91%, respectively. Embryo transfers were performed in 12 cycles except 2 cycles that had no embryos. The number of transferred embryos was 2.3. Two clinical pregnancies were obtained. This scheduled ovarian hyperstimulation was applicable for ICSI with fresh testicular sperm.     

Correlation between the motility of frozen–thawed epididymal spermatozoa and the outcome of intracytoplasmic sperm injection

The purpose of this study was to investigate if the outcome of ICSI was influenced by epididymal sperm motility in frozen-thawed specimens. A total of 18 ICSI treatment cycles using spermatozoa retrieved by microsurgical epididymal sperm aspiration (MESA) were analysed retrospectively. Cryopreservation of epididymal spermatozoa was performed when enough epididymal aspirates were collected. Sixty-nine out of 126 oocytes injected with spermatozoa retrieved by MESA were fertilized, giving a fertilization rate of 54.8%. Out of 18 embryo transfer cycles, 6 (33.3%) achieved pregnancies. Fresh epididymal spermatozoa were used in 5 cycles while frozen-thawed epididymal spermatozoa were used in 13 cycles for ICSI. The fertilization rates were 68.6% (35/51) in the former group and 45.3% (34/75) in the latter group, respectively. There was a significant difference between the two groups (p < 0.05). In ICSI treatments using fresh epididymal spermatozoa, the cells used for injection were all motile. However, motile epididymal spermatozoa could be used in only five ICSI treatment cycles after freeze-thawing. In 6 cycles, only immotile sperm were used for injection of frozen-thawed spermatozoa. The fertilization rate in each group was 68.4% (13/19) and 31.6% (12/38), respectively. There was a significant difference between these groups (p < 0.01). These results indicate that the outcome of ICSI was influenced by sperm motility in frozen-thawed epididymal specimens. When no sperm motility could be recovered after freeze-thawing even with chemical treatments, consideration should be given to retrieving fresh epididymal spermatozoa again to achieve a better fertilization rate in such patients.     

Comparison of pregnancy and neonatal outcomes of intracytoplasmic sperm injection performed with frozen versus fresh testicular sperm

BackgroundIt remains controversial whether there is a difference in the prognosis of intracytoplasmic sperm injection (ICSI) using frozen or fresh testicular sperm in patients with obstructive azoospermia (OA). Moreover, in the available studies, few have tracked neonatal outcomes. This study aimed to compare the pregnancy and neonatal outcomes of ICSI using cryopreserved sperm versus fresh sperm collected by testicular sperm aspiration (TESA).MethodsA total of 317 OA patients treated with ICSI in a university affiliated hospital from January 2016 to December 2020 were included in this study. The participants were divided into two groups according to the type of sperm used for ICSI: frozen sperm group (n=154) and fresh sperm group (n=163). The pregnancy and neonatal outcomes of the two groups were compared.ResultsThe data produced by this study showed no significant statistical difference in the 2 pronuclei (2PN) fertilization rate, 2PN cleavage rate, high-quality blastocyst rate, and the average number of transferred embryos in the frozen and fresh sperm groups. Similarly, no difference was found in implantation rate, clinical pregnancy rate, multiple pregnancy rate, miscarriage rate, premature delivery rate, live birth rate, and gender ratio at birth (P>0.05). The average newborn birth weight was similar in both groups (2,932.61±728.40 vs. 3,100.32±515.64 g, respectively) (P>0.05). A higher incidence of low birthweight (LBW) newborns was found in the frozen sperm group (20.91% vs. 8.49%) (P<0.05). Multiple logistic regression analysis showed that LBW is related to single or twin pregnancies (P<0.01), but not sperm (frozen or fresh) (P>0.05). We further analyzed the twin and single pregnancies in the two groups separately, and found that the incidences of LBW were both similar (P>0.05). There was no difference in the Apgar scores at 1 min and 5 min after birth between the two groups (P>0.05).ConclusionsThe use of frozen testicular sperm by TESA was efficient for men with OA. There were similar pregnancy and neonatal outcomes following TESA-ICSI using frozen or fresh sperm in this retrospective study. Prospective investigations are needed for further validation.     

Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.     

Fertilisation and pregnancy outcome after ICSI in globozoospermic patients without assisted oocyte activation

The successful outcome of intracytoplasmic sperm injection (ICSI) with globozoospermic sperm and non-activated oocytes is reported. Three couples underwent ICSI treatment and two of the patients were siblings. Forty-four non-activated oocytes were injected, 26 oocytes fertilised normally and 17 good quality embryos were obtained. Six embryo transfers were carried out, three with fresh embryos and three with frozen-thawed embryos. Three pregnancies resulted from the fresh embryo transfers and additionally two pregnancies were obtained after the transfer of frozen-thawed embryos. Two healthy babies were born. One twin pregnancy is ongoing. Our case reports demonstrate that in some ICSI attempts undertaken with globozoospermic sperm cells from two of our patients, high fertilisation rates, pregnancies and live births can be achieved, without artificially activated oocytes. Our data also suggest that in some cases, round-headed spermatozoa lack the capacity to activate the oocyte. Therefore, it cannot be excluded that artificial oocyte activation could be of help in globozoospermic patients with complete fertilisation failure.     

The number of spermatozoa collected with testicular sperm extraction is a novel predictor of intracytoplasmic sperm injection outcome in non-obstructive azoospermic patients

The purpose of this study was to determine the relationships between monitors of spermatogenesis and predictors of the intracytoplasmic sperm injection (ICSI) outcome in patients with non-obstructive azoospermia (NOA) undergoing testicular sperm extraction (TESE). Seventy-nine patients with NOA (mean age: 43.6±5.2 years), each of whom yielded (97 000±3040) spermatozoa with conventional TESE, were considered in our analysis. Their partners (mean age: 35.8±5.1 years) underwent a total of 184 ICSI cycles; 632 oocytes were collected, 221 oocytes were injected, 141 oocytes were fertilized, 121 embryos were obtained, 110 embryos were transferred, 14 clinical pregnancies were achieved and only one miscarriage occurred. Multivariate regression analysis indicated relationships between the percentage of fertilized oocytes, transferred embryos and clinical pregnancies with the following variable values: female partner''s age, number of spermatozoa collected, testicular volume, male partner''s levels of follicle stimulating hormone (FSH), number of oocytes collected, number of oocytes injected and number of ICSI cycles. A significant inverse relationship was found between female partner''s age or male partner''s FSH levels and biochemical pregnancies. A significant direct relationship emerged between the number of ICSI cycles and the percentage of oocytes fertilized, embryos transferred and biochemical pregnancies, and between the number of spermatozoa collected per testicular biopsy and biochemical pregnancies. The number of spermatozoa was positively linked to the number of clinical pregnancies, independent of the number of ICSI cycles and the number of oocytes collected/injected. The number of spermatozoa collected, FSH level and testicular volume are monitors of spermatogenesis linked to ICSI success.     

Effect on clinical outcome of the interval between collection of epididymal and testicular spermatozoa and intracytoplasmic sperm injection in obstructive azoospermia

We wished to determine whether the interval between surgical retrieval of epididymal and testicular spermatozoa in obstructive azoospermia and their subsequent use in intracytoplasmic sperm injection (ICSI) has an effect on their fertilizing capacity and pregnancy rates in patients undergoing ICSI. This was a retrospective review of 164 consecutive cycles of ICSI in partners of men undergoing surgical sperm retrieval for obstructive azoospermia. Seventy-three cycles used fresh testicular spermatozoa; in 35 cycles ICSI was performed within 4 hours of sperm retrieval, and in 38 cycles spermatozoa were incubated overnight before ICSI. Epididymal spermatozoa were used in 29 cycles; 22 cases within 4 hours of retrieval and 7 cases following overnight culture. Cyropreserved testicular and epididymal spermatozoa were used in 42 and 20 ICSI cycles, respectively. Fertilization and clinical pregnancy rates were calculated for each treatment group. Fertilization rates for epididymal spermatozoa were 67% at 4 hours, 56% at 24 hours, and 63% for cryopreserved spermatozoa (P =.52). Fertilization rates for testicular spermatozoa were 63% at 4 hours, 71% at 24 hours, and 60% for cryopreserved spermatozoa (P =.16). Unlike testicular spermatozoa, cryopreserved epididymal spermatozoa showed a significant increase in clinical pregnancy rates with cryopreservation, with rates of 4 of 22, 1 of 7, and 10 of 20 at 4 hours, 24 hours, and cryopreservation, respectively (P =.049). This study confirms that fertilization and pregnancy rates following ICSI with motile spermatozoa are unaffected by the duration between surgical retrieval of spermatozoa and their injection into oocytes. It also demonstrates that of all treatment modalities, the use of frozen epididymal spermatozoa was associated with the greatest pregnancy rates.     

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection （ICSI） using ejaculated and testicular spermatozoa

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.     

Sperm morphometry: a tool for detecting biophysical changes associated with viability in cryopreserved bovine spermatozoa

The aim of this study was to determine whether computerised sperm head morphometric analysis can be used as a diagnostic tool for detecting biophysical changes associated with sperm viability in frozen–thawed bovine spermatozoa. Ejaculates from five bulls (4 ejaculates/bull) were pooled and processed for computerised morphometric analysis, and SYBR‐14 green/ethidium homodimer‐1 fluorescence‐based live/dead viability assay was used simultaneously to confirm the viability index of frozen–thawed spermatozoa. Sperm samples were assigned to three experimental groups. The first group was enriched in live spermatozoa (after a double Percoll selection), the second group was enriched in dead spermatozoa (after a refreeze–thaw procedure), and the last group was a 50 : 50 pool of live/dead spermatozoa (from first and second group samples). There were significant differences (P < 0.001) related to sperm morphometric dimensional parameters among the three groups analysed, being the lowest overall sperm head dimension found in the second (dead spermatozoa) group. In conclusion, sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in frozen–thawed bovine spermatozoa.     

Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle

In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.     

Schedule to inject in vitro matured oocytes may increase pregnancy after intracytoplasmic sperm injection

To ascertain the value of using immature oocytes in an intracytoplasmic sperm injection (ICSI) program, the authors designed a schedule, at 5 p.m. on day 1 (the day of oocyte retrieval) and at 8 a.m. and 2 p.m. on day 2, to recognize and inject the in vitro matured (IVM) oocytes. For the 1,166 oocytes retrieved in 107 ICSI cycles, 128 (11.0%) were at the stage of metaphase I (MI) and 113 (9.7%) at germinal vesicle. Routine ICSI for metaphase 11 oocytes was performed at 2 p.m. on day 1 (initial ICSI). In culture medium of human tubal fluid with 15% maternal serum, 85.1% (205/241) immature oocytes progressed to maturation in which 16.4% (21/128) of MI oocytes matured at 5 p.m. of day 1. The rate of normal fertilization for IVM oocytes (58.5%) was not significantly different from that of initial ICSI (64.0%). One patient received a transfer of two fertilized IVM oocytes alone that were injected at 5 p.m. of day 1, maturing from the MI stage, and achieved a normal pregnancy. The fertilized IVM oocytes were replaced along with the embryos from initial ICSI for 40 cycles that led to 14 (35%) clinical pregnancies. In 43 fertilized IVM oocytes donated for research, we observed that cleavage (95.3%) to the 2- to 4-cell stage was not distinct from that of initial ICSI (94.6%); however, the percentage of embryos of grade I and II morphology was significantly smaller (24.4% vs. 62.5%). Only five (11.6%) developed to blastocysts in vitro. Twenty-one fertilized IVM oocytes were frozen for future transfer. A schedule to inject IVM oocytes in ICSI cycles may generate more accessible embryos for fresh transfer or cryopreservation to increase the chance of pregnancy, although the embryo quality was relatively poor.     

单精子卵细胞质内注射治疗梗阻性无精子症

目的:总结单精子卵细胞质内注射治疗梗阻性无精子症的诊疗经验。方法:回顾总结2006年1月～2008年12月间107例梗阻性无精子症病例ICSI助孕资料,比较先天性输精管缺如组与非先天性输精管缺如组之间受精率、卵裂率以及妊娠率的差异。结果:107例梗阻性无精子症病例ICSI助孕中共行单精子卵细胞质内注射949枚卵子,形成受精卵678枚(受精率71.4%),获得胚胎卵裂605枚(卵裂率89.2%),临床妊娠44例,临床妊娠率41.1%。其中先天性输精管缺如49例,行单精子卵细胞质内注射442枚卵子,形成受精卵308枚(受精率69.6%),获得胚胎卵裂279枚(卵裂率90.6%),临床妊娠27例,临床妊娠率55.1%;炎症或手术等原因引起的梗阻性无精子症58例,行单精子卵细胞质内注射507枚卵子,形成受精卵370枚(受精率72.9%),获得胚胎卵裂326枚(卵裂率88.1%),临床妊娠17例,临床妊娠率29.3%。两组比较受精率、卵裂率无统计学差异(P>0.05),临床妊娠率有统计学差异(P<0.01)。结论:采用经皮附睾或睾丸穿刺抽吸精子结合ICSI技术助孕是治疗梗阻性无精子症的安全有效方法。先天性输精管缺如较其它原因所导致的梗阻性无精子症有更高的临床妊娠率。炎症或手术等原因除引起精道梗阻外也可能影响精子的质量,导致胚胎发育潜能下降。     

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation

Summary.  Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG_2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.     

Analysis of the outcome of intracytoplasmic sperm injection using fresh or frozen sperm

Study Type – Diagnostic (retrospective cohort)
Level of Evidence 2b What’s known on the subject? and What does the study add? The results of ICSI using fresh or frozen sperm on the site of sperm retrieval remains controversial with respect to outcome. The results of this study showed no difference in outcome using ICSI either with respect to the site of retrieval or whether the sperm used was fresh or frozen. It also showed that the outcome of ICSI is not related to the underlying cause of the azoospermia.

OBJECTIVES

? To compare the outcome of first‐attempt intracytoplasmic sperm injection (ICSI) ICSI–embryo transfer (ET) cycles using frozen‐thawed testicular sperm (FTTS), fresh testicular sperm (FTS), frozen‐thawed epididymal sperm (FTES) and fresh epididymal sperm (FES) so as to determine which of these has the most successful ICSI outcome with respect to fertilization rate (FR), pregnancy rate (PR) and birth rate. ? To assess the outcomes according to the underlying aetiology of azoospermia.

PATIENTS AND METHODS

? The records of 493 patients undergoing first‐attempt ICSI between 1993 and 2008 were reviewed retrospectively. FTS was used in 112 cycles, FTTS in 43 cycles, FES in 279 cycles, and FTES in 59 cycles. ? Within each group, the aetiology of the azoospermia was recorded according to history, clinical examination and histological analysis (n= 316). ? The FR, clinical PR and delivery rate were calculated for each group with respect to the type of sperm retrieval used.

RESULTS

? Analysis of the data showed no significant differences between any of the four groups in the FR, PR or delivery rate (P > 0.05). ? There were no significant differences seen between fresh sperm (FTS and FES) and frozen sperm (FTTS and FTES) or between epididymal sperm (FES and FTES) and testicular sperm (FTS and FTTS) in any of the outcomes measured (P > 0.05). However, sub‐set analysis showed a statistically higher FR and PR for FTTS over fresh sperm. ? When comparing aetiologies, there was no significant difference in the FR, clinical PR and delivery rate between obstructive azoospermia (OA) and non‐obstructive azoospermia (NOA) groups. However, sub‐set analysis showed a higher PR and birth rate for FTTS over fresh sperm in both OA and NOA groups.

CONCLUSIONS

? The results of the present study suggest that using frozen sperm in ICSI cycles is a reliable and favourable method with the same outcome as fresh sperm. ? Testicular and epididymal sperm have similar ICSI outcomes for both fresh and frozen samples. However, results suggest a tendency for higher PRs and birth rates for frozen than for fresh testicular sperm in both OA and NOA aetiologies. ? The aetiology of azoospermia does not significantly affect the outcome of first‐attempt ICSI. The higher rates in the frozen groups suggest that these patients have had better quality semen when they were initially harvested and frozen.     

Testicular sperm retrieval: What should we expect from the fresh and subsequent cryopreserved sperm injection?

We sought to compare ICSI outcomes of cycle using fresh versus thawed TESE spermatozoa obtained during the previous fresh TESE. All consecutive couples undergoing ICSI cycles using fresh TESE spermatozoa, followed by ICSI cycle using cryopreserved sperm remaining from the previous fresh TESE procedure were included. Ovarian stimulation (OS)/laboratory variables and cycle outcome were assessed and compared between those utilising fresh versus thawed TESE spermatozoa. Seventy-five couples were evaluated, with no in-between groups differences in OS nor embryological variables. While implantation and LBR per embryo transfer were nonsignificantly higher in the frozen as compared to the fresh TESE, there was a trend towards higher LBRs per patient in the frozen TESE group. The cumulative miscarriage rate (4% versus 14.7%, p < .022 respectively) was significantly lower and the cumulative LBR (34.7% versus 16%, p < .007 respectively) was significantly higher using frozen TESE spermatozoa. Moreover, significantly higher proportion of frozen TESE sperm samples used pentoxifylline to enhance sperm motility. In conclusion, the results of ICSI cycles using frozen TESE spermatozoa are as good, or even better than using fresh TESE spermatozoa. Further studies are required to explore the factors responsible for the improved ICSI outcome, while using frozen versus fresh TESE sperm samples.     

经皮附睾穿刺取精精子冷冻复苏后卵细胞胞质内单精子注射治疗无精子症的初步研究

目的:回顾性分析27例无精子症患者经皮附睾穿刺取精术(PESA)所获精子冷冻复苏后行卵细胞胞质内单精子注射(ICSI)治疗后的效果及妊娠结局。方法:将诊断性附睾穿刺以及PESA治疗周期ICSI后所剩余活精子以常规方法加以冷冻,将复苏后找到了足量活精子并行ICSI的病例归为冻精组,而采用新鲜PESA活精子ICSI的病例则归为对照组。比较冻精组与对照组的受精率、种植率、临床妊娠率,同时分析两组间的妊娠并发症、新生儿出生及畸形等情况。结果:冻精组15个周期、对照组100个周期分别注射MⅡ期成熟卵子163、1 157个,受精率冻精组显著高于对照组(84.05%vs73.29%,P<0.05),种植率、临床妊娠率则两组间差异无显著性(23.07%vs15.73%;53.33%vs37.00%,P>0.05),新生儿出生体重差异亦无显著性(P>0.05)。冻精组共妊娠8例,已分娩5例,继续妊娠3例。对照组妊娠37例,已分娩30例,1例死胎;继续妊娠3例;流产4例。两组均未出现重大的妊娠并发症及新生儿畸形。结论:采用PESA冷冻精子ICSI是治疗男性无精子症的一种经济、有效、安全的方法;但PESA冻精复苏率有待于进一步提高。     

Various physical stress factors on rat sperm motility, integrity of acrosome, and plasma membrane

The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 x g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 x g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 x g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.     

Tales of the tail and sperm head aches: changing concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail

This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail.