Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.     

Dizygotic twin pregnancy after intracytoplasmic sperm injection of 1 day old unfertilized oocytes

We report on a case where late intracytoplasmic sperm injection (ICSI) on unfertilized oocytes after standard in-vitro fertilization (IVF) cycles resulted in a dizygotic twin pregnancy. Fifteen oocytes were harvested from a patient with a history of salpingotomy. After a single cycle of IVF, only one oocyte showed two pronuclei. Subsequently ICSI was performed on six unfertilized metaphase II oocytes, and three of these oocytes showed two pronuclei. Three fertilized embryos were transferred (two derived from ICSI and one from IVF). A normal twin pregnancy resulted, and after delivery of two healthy boys the twins were confirmed to be dizygotic by DNA analysis of several loci. We conclude that at least one of the embryos was derived from the reinsemination by 'second day ICSI'.     

Development of embryos produced by intracytoplasmic sperm injection of cat oocytes

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.     

Intracytoplasmic sperm injection as a routine indication in low responder patients

The aim of this study was to determine if a low response to gonadotrophin stimulation could be considered as an indication for intracytoplasmic sperm injection (ICSI). This prospective study included a total of 96 non-male infertile couples with six or fewer retrieved oocytes, who underwent 104 in-vitro fertilization (IVF) cycles between January 1996 and April 1997. They were randomly divided into two groups for fertilization, one by IVF and the other by ICSI. Groups were compared in terms of fertilization rates, fertilization failure, embryo quality, embryos transferred and reproductive outcome. ICSI provided similar fertilization rates per inseminated oocyte (77.7 versus 70.2%) and per obtained oocyte (56.5 versus 58.8%) as IVF. Furthermore, equal numbers (2.2 versus 2.5) and quality of embryos were obtained and comparable pregnancy (21.1 versus 17.3%) and implantation (14.0 versus 11.1%) rates. Neither the number of retrieved oocytes, nor patient age was relevant for the fertilization rates obtained with both techniques. The number of cases with complete fertilization failure was similar in both procedures. We conclude that the technique of fertilization is not related to the reproductive outcome of low responders, and the routine use of ICSI is not indicated.     

Relationship between human in-vitro fertilization and intracytoplasmic sperm injection and the zona-free hamster egg penetration test

The zona-free hamster egg penetration test (HEPT) is widely used for evaluating the fertilizing ability of human spermatozoa. However, the relationship between the HEPT and microassisted fertilization has yet to be determined. To evaluate the efficiency of HEPT in selecting the most appropriate method of in-vitro fertilization (IVF), including intracytoplasmic sperm injection (ICSI) in couples with male factor infertility, clinical laboratory data was analysed retrospectively. The patients were divided into groups according to the sperm penetration index as determined by the HEPT: group A (sperm penetration index = 0), group B (sperm penetration index < 15) and group C (sperm penetration index > or = 15). A total of 405 oocytes were collected and inseminated by conventional methods in 69 couples with male factor infertility. In all, 31 out of 148 (20.9%) oocytes fertilized in group A; 35 out of 117 (29.9%) in group B; and 73 of 140 (52.1%) in group C. The clinical pregnancy rates per transfer in groups A, B and C were 0% (0/13), 0% (0/14) and 25.9% (7/27) respectively. Both the fertilization rate and pregnancy rate in group C was significantly higher than in groups A and B. ICSI was carried out in a total of 57 couples and 334 oocytes in metaphase II stage were manipulated. The normal fertilization (2 pronuclear) rate per oocyte was 65.6 +/- 26.0% (mean +/- SD). Out of 127 oocytes, 76 (59.8%) fertilized in group A, 57 out of 87 oocytes (65.5%) in group B and 86 out of 120 oocytes (71.7%) in group C. Of the 56 transfers, 17 clinical pregnancies were obtained, giving an average pregnancy rate of 30.4% per transfer. The clinical pregnancy rates per transfer in groups A, B and C were 17.4% (4/23), 40.0% (4/10) and 39.1% (9/23) respectively. No significant differences were observed in the fertilization rates or in the pregnancy rates between the three groups. In addition, there were no differences in the fertilization and pregnancy rates between the ICSI and IVF patients in group C. These findings suggest that the results of the HEPT are well correlated with the fertilizing ability of human spermatozoa in the patients treated by conventional IVF. Couples suffering from male factor infertility with a sperm penetration index of < 15 (as determined by HEPT) should consider treatment with ICSI, while those with a sperm penetration index of > or = 15 should attempt conventional IVF.     

Timing of oocyte activation, pronucleus formation and cleavage in humans after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa

In the first study, we evaluated 101 oocytes [2, 4, 6, 8, 16, 18 and 20 h after intracytoplasmic sperm injection (ICSI)] that had been microinjected with testicular spermatozoa. Of the 70 normally fertilized oocytes (69%) 30 (43%) had two pronuclei by 6 h after ICSI. Fifty-one (73%) by 8 h, 69 (99%) by 16 h and four of them by 20 h cleaved to the 2-cell stage. In the second study, 95 cumulus-corona-oocyte complexes (CCOC) were divided into two groups. Forty-seven CCOC were inseminated by conventional in-vitro fertilization (IVF) and 40 metaphase-II oocytes by ICSI. Oocytes were evaluated at 2, 4, 6 (only after ICSI), 8, 10, 12, 18, 20, 22, 24, 26, 28 and 30 h after both ICSI and IVF. After IVF, 35 oocytes were fertilized normally (75%), four of which (11%) had two pronuclei by 8 h, 11 (31%) by 10 h, 27 (77%) by 12 h and 35 (100%) by 14 h. The first cleavages had occurred by 24 h after insemination (four oocytes, 11%). After ICSI, 34 oocytes were fertilized normally (79%), 13 of which (38%) had two pronuclei by 6 h, 27 (79%) by 8 h and 32 (94%) by 10 h. Three oocytes cleaved by 20 h after microinjection (9%) and 19 by 24 h (56%). Pronuclei developed asynchronously in six oocytes after ICSI (18%) as opposed to 16 oocytes after IVF (46%). The results of this study suggest that the timing of pronuclear formation is no different when a testicular spermatozoon is microinjected into the oocytes from when an ejaculated spermatozoon is injected. Secondly, pronuclear development and first cleavage generally take place 4 h sooner after ICSI than after IVF. On the other hand, a higher proportion of oocytes develop two pronuclei asynchronously after IVF than after ICSI.     

An interview with Sue Hall Esleck. Interview by Connie R. Curran

The present report covers the results of a 38-month period in which 2853 consecutive intracytoplasmic sperm injection (ICSI) cycles were performed in 1953 couples. These couples were afflicted with male factor infertility and had at least one previous failed conventional in vitro fertilization (IVF) treatment cycle. In other couples, the husband had semen parameters incompatible with conventional IVF or suffered from excretory or secretory azoospermia where it was possible to recover spermatozoa by microsurgical epididymal sperm aspiration (mesa) or by testicular sperm extraction (tese) procedure. Overall, the 2-PN fertilization rate was 62% per retrieved metaphase II oocyte and 70% per successfully injected metaphase II oocyte. Embryo transfer was performed in 91% of started cycles. The cumulative pregnancy rate (positive HCG) was 34% per started ICSI treatment and 37% per embryo transfer.     

Sperm-induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic changes following intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has increased the potential of the assisted reproductive technologies to propagate mammalian species and has provided an opportunity for research into cell cycle control and the mechanisms involved in sperm-induced oocyte activation. We have investigated the efficacy of ICSI in the rhesus monkey, the mechanism of fertilization following sperm injection and the cytoskeletal rearrangement that occurs upon oocyte activation. These studies were conducted on mature, and to a lesser extent, immature oocytes. Ejaculated spermatozoa, washed, capacitated and activated before immobilization, were injected into oocytes using conventional ICSI methodology. Sperm injection into mature oocytes induced oocyte activation (19/22; 86%) and pronuclear formation. In contrast, sham-injected oocytes did not activate readily (2/16; 13%). To localize oocyte activation factor(s), spermatozoa were separated mechanically into heads and tails which were then injected individually into mature oocytes. Activation occurred in 87% (20/23) of oocytes receiving heads. After tail injection, a single microtubule aster was nucleated and one pronucleus (PN) was seen in four of 21 oocytes. Intracytoplasmic injection of sperm extract (SE) resulted in oocyte activation at a significantly higher rate than occurred following sham injection (76 versus 13%). Sperm-induced oocyte activation was also evaluated in immature metaphase (MI) oocytes; activation occurred in 46% (12/26) of cases; however, only 8% of the activated oocytes exhibited 2 PN. Finally, beta-tubulin localization in untreated and taxol-treated oocytes was established as a marker for cytoplasmic changes associated with oocyte activation. These results are consistent with the hypothesis that spermatozoa contain an oocyte activating factor(s) which is primarily localized in the sperm head. Moreover, an activation response is limited to mature oocytes and is accompanied by cytoskeletal changes analogous to those seen following conventional fertilization.     

Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection

During the intracytoplasmic sperm injection (ICSI) procedure, the collected oocytes are incubated until just before ICSI. The ideal preincubation time of oocytes was investigated in 544 treatment cycles. Oocyte retrieval was carried out 35 h after human chorionic gonadotrophin administration. Oocytes were cultured for between 1 and 11 h before ICSI. Embryo transfer was performed 48 h after oocyte collection. The survival, fertilization and cleavage rates of injected oocytes indicated no statistically significant differences between oocytes preincubated for different lengths of time. The proportion of good-quality embryos (grades 1 and 2) was lower at 9-11 h of preincubation time than for all the other preincubation times (P < 0.001). No statistically significant differences were detected in the pregnancy rate between each group (mean: 15.9%), although the pregnancy rate at 9-11 h of preincubation time appeared to be low (7.7%). These results suggest that the oocyte retained sufficient potential for fertilization between 1 and 9 h after oocyte collection in ICSI. For the researchers who practise more complex ICSI procedures than IVF, it would be convenient to be able to perform ICSI at any time between 1 and 9 h after oocyte collection.     

Oocyte maturation and intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI), treatment of severe male infertility allows an accurate evaluation of oocyte maturity at recovery after corona-cell removal. In cycles comprising a GnRH analog desensitization and a stimulation by hMG or FSH, 12% of oocytes aspirated from follicles (> 14 mm), 34 hours post-hCG are still immature, in prophase or metaphase 1. They are able to achieve meiosis in vitro in 66% of the cases and will be fertilized (2 PN) by ICSI in 51% of the cases as the in vivo mature oocytes of the same cohort. Nevertheless, the quality of cytoplasmic maturation and consequently of embryonic viability remains to be assessed as there still are few pregnancies arising from in vitro matured oocytes. ICSI also represents the only way to obtain normal fertilization in some exceptional but observed anomalies of oocyte maturation, particularly when there is a lack of zona reaction leading to repetitive polyspermy in conventional IVF.     

Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report

OBJECTIVE: To report a normal pregnancy and the delivery of a healthy child after the combination of in vitro maturation of germinal-vesicle stage oocytes and intracytoplasmic sperm injection (ICSI) in a patient. SETTING: Procedures were performed in a tertiary IVF center coupled with an institutional research environment. MAIN OUTCOME MEASURES: Maturation rate of immature oocytes after in vitro maturation and intactness, fertilization, and developmental rates of oocytes after microinjection. RESULTS: Nine of 14 germinal-vesicle stage oocytes matured to the metaphase II stage after 30 hours of in vitro culture (64%). Seven of eight injected and intact oocytes fertilized normally (78%) and five of them cleaved with < 20% fragmentation (71%). Four embryos were transferred and a singleton pregnancy was obtained that ended in the delivery of a healthy child. CONCLUSION: In vitro maturation of immature oocytes together with ICSI can result in normal fertilization, embryo development, pregnancy, and the delivery of healthy child.     

Possible mechanisms involved in apoptosis of colon tumor cell lines induced by deoxycholic acid, short-chain fatty acids, and their mixtures

OBJECTIVE: To study the effects of cryopreservation on the quality of human testicular spermatozoa and the efficiency of intracytoplasmic sperm injection (ICSI) with frozen-thawed testicular sperm into metaphase II oocytes in vitro-matured from the germinal-vesicle stage oocyte. DESIGN: Preclinical freezing study on supernumarary testicular spermatozoa after ICSI. SETTING: Tertiary IVF center coupled with an institutional research environment. PATIENT(S): Twenty-nine patients undergoing excisional testicular biopsy for ICSI. INTERVENTION(S): Isolated testicular spermatozoa were cryopreserved and thawed; frozen-thawed motile testicular spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Prefreezing and post-thawing motility and viability, survival rate, fertilization rate, cleavage rate, and embryo quality after ICSI. RESULT(S): Mean percentage motility decreased from 21% before freezing to 6% after thawing. Vitality was impaired to a similar extent, decreasing from 68% to 22% (32% recovery rate). Injection of frozen-thawed testicular spermatozoa into in vitro-matured oocytes resulted in a fertilization rate of 50.9%. Cleavage rate was severely impaired. Half of the fertilized oocytes became arrested in the one-cell stage. CONCLUSION(S): Despite the low quality of the fresh testicular spermatozoa, a high percentage of prepared testicular sperm fractions showed survival and motility after the freezing and thawing process. Injection of frozen-thawed testicular sperm into matured oocytes resulted in fertilization rates comparable with these with fresh testicular sperm, but cleavage rates were severely impaired, which might be due to source of oocytes used for ICSI.     

Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection

OBJECTIVE: To compare the survival rate and pregnancy rate (PR) of embryos from intracytoplasmic sperm injection (ICSI) or conventional IVF, which were cryopreserved at the pronuclear stage in cycles where fresh transfer was deferred. DESIGN: Comparative observational study. SETTING: University-associated IVF center. PATIENT(S): Ninety-nine patients who deferred ET and had all their embryos cryopreserved at the pronuclear stage after 153 oocyte retrievals. Thirty-nine patients had their oocytes inseminated by ICSI and 60 patients had conventional IVF insemination. INTERVENTION(S): All embryos were frozen-thawed at the two pronuclear stage and allowed to cleave for 2 days before transfer. MAIN OUTCOME MEASURE(S): Survival rate (morphologically intact after thaw), cleavage rate (cleaved by time of transfer), and the clinical PR after frozen ET. RESULT(S): In the ICSI group, 205 embryos were thawed for use in 57 frozen ETs; in the IVF group, there were 527 embryos thawed for use in 149 frozen ETs. There was no significant difference in any of the outcome measures by insemination method: survival rates (ICSI, 93.2%; IVF, 94.8%); cleavage rates (ICSI, 95.2%; IVF, 94.7%), and clinical PR (ICSI, 14.0%; IVF, 17.4%). CONCLUSION(S): Pronuclear embryos resulting from ICSI can be cryopreserved successfully, thawed, and the survival rate and PR are comparable to conventional IVF.     

Implications of complete fertilization failure after intracytoplasmic sperm injection for subsequent fertilization and reproductive outcome

With the introduction of intracytoplasmic sperm injection (ICSI), couples with severe male factor infertility have achieved fertilization and clinical pregnancy rates comparable to other in-vitro fertilization (IVF) patients. However, failure of fertilization still occurs in some patients despite the utilization of microsurgical sperm injection techniques. How such fertilization failure after ICSI might impact later ICSI treatment(s) is unknown. In this investigation, couples with complete fertilization failure after ICSI treated from August 1993 to August 1996 were identified (index cycle, n = 21). Additionally, fertilization data from any previous or subsequent infertility treatments were evaluated. Seven patients (33%) had at least one IVF treatment before the index cycle, although no deliveries occurred. Of patients with complete fertilization failure in the index cycle, 48% (n = 10) underwent at least one subsequent ICSI cycle which proceeded to oocyte retrieval. The remainder (n = 11) elected to discontinue treatment. Although six subsequent cycles were cancelled due to poor follicular response (< or = 2 mature oocytes), all patients electing to continue treatment eventually achieved a subsequent embryo transfer. The clinical pregnancy rate per transfer was 45.4% for this group; the delivery and ongoing pregnancy rate per transfer was 36.3%. Review of semen parameters, superovulation characteristics or other clinical parameters during the three study cycles (pre-index, index, and post-index) was not prognostic of fertilization success or reproductive outcomes in later treatments. Fertilization failure with ICSI therefore could not be predicted by prior cycle performance, although total immotility of spermatozoa at time of oocyte retrieval, total teratozoospermia, and low oocyte yield were common characteristics of couples experiencing complete fertilization failure with ICSI. These findings suggest that fertilization failure in one ICSI cycle does not preclude successful fertilization and delivery in a later ICSI treatment.     

Factors influencing sperm-zona pellucida binding in vitro using the intact zona model

The zona pellucida binding assay assesses the ability of spermatozoa to bind to the zona pellucida. The present study investigated the influence of: (i) prior oocyte exposure to spermatozoa on subsequent sperm-zona pellucida binding in vitro; and (ii) cryopreservation of oocytes. Only oocytes obtained from fertile donors were used and the binding capacity of non-inseminated, cryopreserved oocytes was compared with both inseminated/unfertilized, cryopreserved oocytes and inseminated/unfertilized, non-cryopreserved oocytes recovered from in-vitro fertilization cultures on sperm-zona pellucida binding using an intact zona model. There was no statistically significant difference in sperm-zona binding between non-inseminated, cryopreserved oocytes (range 9.6-23.2), inseminated/unfertilized, cryopreserved oocytes (range 15.0-16.0) and inseminated/ unfertilized, non-cryopreserved oocytes (range 3.3-23.0). The coefficient of variation for sperm binding to all oocyte groups was very large (range 37-121%). We conclude that neither prior exposure of human oocytes to human spermatozoa nor cryopreservation of human oocytes influences the subsequent binding of a different population of spermatozoa to the zona pellucida. However, large oocyte-to-oocyte variation of sperm-zona binding may diminish the usefulness of this assay in clinical practice and as a research tool.     

Quantification of ribonucleotide reductase expression in wild-type and hydroxyurea-resistant cell lines employing in situ reverse transcriptase polymerase chain reaction and a computerized image analysis system

PURPOSE: Because the availability of prophase oocytes for zona binding testing is limited, we compared sperm binding to the zona of failed-fertilized intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) oocytes after incubation in a standard IVF medium and a specially composed binding improvement medium. METHODS: Semen samples from nine patients and nine fertile donors were separated in parallel by the standard swim-up method in both media. Subsequently, hemizona assays were performed with prophase, failed-fertilized ICSI and IVF oocytes. RESULTS: Sperm separation resulted in a significantly higher sperm count (P < 0.01) and progressive motility (P = 0.018) in binding improvement medium. Moreover, spermatozoa coincubated with hemizonae (prophase, failed-fertilized ICSI and IVF oocytes) in binding improvement medium bound significantly more to hemizonae than in the controls (P < 0.01). However, the hemizona index did not differ. CONCLUSIONS: Thus, the limited number of human zonae can be increased by the use of oocytes that failed to fertilize during ICSI or IVF. This will lead to a qualitative improvement of the diagnostic spectrum in male-factor infertility.     

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.     

Are there indications for intra-cytoplasmic sperm injection in female infertility?

ICSI for oocyte pathology may represent the solution in case of zona pellucida anomalies, deficiency of the oolemma fusion ability or absence of cortical reaction. These indications remain scarce and difficult to be displayed. A link could be shown by some authors between some oocyte morphologic anomalies and IVF fertilization failures which are overcome by ICSI. Other dysmorphic oocytes are associated with an impairment in cytoplasm function which results in a poor embryonic viability on which ICSI has no effect. When few oocytes are recovered (< or = 5) and the semen characteristics are not optimal, ICSI may rectified the decrease in fertilization rates obtained after classical insemination. Lastly, ICSI will become increasingly used in case of oocyte in-vitro maturation or freezing instead of classical IVF which is less efficient.     

Initial experience with the MESA and TESE techniques in the treatment of infertile men with azoospermia

The authors evaluate their initial experience and results of the technique of microsurgical aspiration of epididymal sperm (MESA) and extraction of testicular sperm (TESE) combined with intracytoplasmic sperm injection into the oocyte (ICSI) in men with azoospermia. The above methods were used in 16 patients with repeatedly assessed azoospermia. Epididymal sperm was obtained in six instances, testicular sperm in 10 instances. With epididymal sperm after ICSI 50% fertilization was achieved after ICSI, with testicular sperm 51% fertilization of oocytes.     

A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule

OBJECTIVE: To determine whether spermatozoa, located in the seminiferous tubules obtained by needle puncture testicular biopsy, could be cryopreserved successfully within the tubules and subsequently be used for in oocyte fertilization via intracytoplasmic sperm injection (ICSI) after the spermatozoa were removed from the thawed tubules. DESIGN: Clinical case series. SETTING: Private IVF unit. PATIENT(S): Six azoospermic patients (four obstructive, two maturation arrest). MAIN OUTCOME MEASURE(S): Survival rate of thawed spermatozoa, fertilization rate of oocytes after ICSI with spermatozoa extracted from thawed tubules and pregnancies. RESULT(S): All six patients had adequate motile spermatozoa extracted from the thawed tubule sections, and all patients achieved fertilization via ICSI with their partner's eggs. The fertilization rate was 46%, compared with 56% obtained in other previous patient cycles using fresh testicular spermatozoa. Three pregnancies resulted from five ETs. CONCLUSION(S): Cryopreservation and subsequent thawing of seminiferous tubules proved to be a simple and successful method for storage of testicular spermatozoa, reducing the need for repetitive testicular biopsies and increasing the likelihood of sperm availability on the day of oocyte retrieval.