Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey

Notwithstanding the thousands of seemingly healthy children born after intracytoplasmic sperm injection (ICSI), it is not yet possible to conclude absolutely that the ICSI procedure might induce some altered development or that the ICSI protocol might not be improved even further. To address this in a clinically relevant system, the developmental potential of rhesus monkey embryos produced by ICSI is reported. Oocytes collected by laparoscopy from gonadotrophin-stimulated fertile females were fertilized by ICSI using spermatozoa obtained from fertile males by electro-ejaculation. Neither sperm immobilization prior to injection nor an additional chemical stimulus were necessary to achieve oocyte activation and pronuclear formation. Survival and activation of the injected oocytes were judged by the extrusion of the second polar body. Successful fertilization was confirmed by the presence of two pronuclei within 12 h post-ICSI. Some oocytes were fixed and processed for the detection of microtubules and chromatin. Fluorescent labelling revealed that by 12 h post-ICSI the male and female pronuclei were closely apposed and eccentrically positioned within a large microtubule aster. ICSI resulted in a 76.6 +/- 14.9% fertilization rate. First cleavage was completed within 24 h post-ICSI. Two-cell ICSI embryos were co-cultured in CMRL medium on a buffalo rat liver cell monolayer until the hatched blastocyst stage. Oocytes collected laparoscopically from stimulated monkeys can be fertilized by ICSI and will complete preimplantation embryo development in vitro demonstrating that the rhesus monkey is an excellent preclinical model for examining and understanding many aspects of human ICSI.     

Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles

From October 1992 to December 1994, 2732 cycles of treatment by intracytoplasmic sperm injection (ICSI) were carried out in couples mainly with severe male-factor infertility. The overall fertilization rate in these 2732 cycles was 71% of intact oocytes. However, in 76 (72 couples) of these cycles, none of the injected oocytes became fertilized, so the total fertilization failure rate was 3% (76/2732 cycles). Details of these 76 cycles were analysed. The results show that total fertilization failure after ICSI may be explained by different factors related to (i) semen characteristics (only immotile or round-headed spermatozoa for ICSI) or (ii) the oocytes (number, abnormal morphology, damage after ICSI). Of 26 couples, 22 achieved fertilization in their subsequent ICSI cycles. In conclusion, total fertilization failure after ICSI for the treatment of severe male-factor infertility was mainly caused by the poor viability of the spermatozoa used for injection; it was also associated with a low number and poor quality of oocytes. Repeated ICSI treatment may be useful or necessary in couples with total fertilization failure.     

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.     

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.     

Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection

The microinjection of completely immotile spermatozoa may impair the outcome of intracytoplasmic sperm injection (ICSI). Eleven couples underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa. Two-pronuclear fertilization ensued in 18 of 145 (12.4%) successfully injected oocytes. None of these cycles resulted in a pregnancy. Nine couples underwent ICSI in subsequent cycles (n = 16). Ejaculated spermatozoa were injected in 15 cycles and testicular spermatozoa in one cycle. In 10 of the 15 cycles, motile spermatozoa were available at the time of injection. Motile testicular spermatozoa could also be injected. In the subsequent cycles, 91 of 176 (51.7%) successfully injected oocytes fertilized normally and four patients became pregnant. In the subsequent cycles where again immotile spermatozoa had to be injected no pregnancies occurred. In four subsequent cycles embryo cryopreservation was carried out. After replacement of two frozen-thawed embryos one additional pregnancy was obtained. In all, five healthy infants were born. It has been ascertained that motile spermatozoa can be detected either in repeated ejaculates or after testicular biopsy. The causes of total asthenozoospermia are variable and the problem is a sporadic rather than a permanent condition.     

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.     

Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia

Testicular or epididymal spermatozoa were obtained for in-vitro fertilization and intracytoplasmic sperm injection (ICSI) in 27 cycles out of 33 (in six men the azoospermia proved to have testicular causes). Testicular needle biopsy carried out in addition to surgical open biopsy proved to be an effective method to obtain spermatozoa for ICSI from patients with obstructive azoospermia. Thus it might be possible to replace scrotal operations by simple needle biopsies. Embryos resulting from ICSI with testicular spermatozoa were used in 19 transfers that resulted in six pregnancies. One pregnancy resulted from six embryo transfers from ICSI after microsurgical-epididymal sperm aspiration (MESA). The normal fertilization rates with testicular (37.3%) and MESA spermatozoa (53.7%) did not differ significantly from each other, but with testicular spermatozoa the rate was significantly lower than that obtained with ejaculated spermatozoa and ICSI (59.7%) in the matched couples. The abnormal fertilization of oocytes with one pronucleus was significantly higher with testicular spermatozoa than with ejaculated spermatozoa in the control couples.     

Three-dimensional low dose gadolinium-enhanced peripheral MR venography

Cases with absolute immotile sperm syndrome are rare, and include the genetic defect of immotile cilia syndrome with the absence of dynein arms in the flagellum. We attempted to increase the percentage of viable spermatozoa to improve the outcome of intracytoplasmic sperm injection (ICSI). Three couples in whom repeated analysis of the male partners indicated 100% sperm immotility underwent an in-vitro fertilization (IVF) procedure in which ICSI was performed. On their first ICSI cycle the males produced a single ejaculation while in their successive ICSI cycles they were requested to repeatedly ejaculate (two to four times) and only the last ejaculation was used. The eosin-Y test was performed on each used sample. Following their first treatment, one couple had one repeated treatment cycle, another had two and the third couple had three repeated treatment cycles. The mean percentages of viable spermatozoa were 41+/-7.4 and 71+/-6.9% in the first and repeated cycles respectively (P < 0.01; t-test). Of the 39 oocytes injected in the first ICSI cycles only one (3%) was normally fertilized (2PN) compared with 41 (48%) of the 85 oocytes injected in the repeated ICSI cycles. One (3%) embryo in the first and 35 (41%) embryos in the repeated ICSI cycles respectively were obtained (P < 0.001), enabling their replacement into the uterine cavity in all the repeated cycles. One woman (in a repeated cycle) conceived a twin pregnancy and delivered two healthy babies. The use of spermatozoa from repeated ejaculation is recommended in men with absolutely immotile spermatozoa so as to obtain significantly better viability and fertilizing capacity.     

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.     

Cytogenetic abnormalities of unfertilized oocytes generated from in-vitro fertilization and intracytoplasmic sperm injection: a double-blind study

In the present study we have assessed the cytogenetic abnormalities of unfertilized oocytes from in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) programmes during a one year period (July 1995 to July 1996) with the cytogenetic analysis being carried out in a double-blind manner. A total of 88 unfertilized ICSI and 85 unfertilized IVF oocytes were used for the study and of these 51 and 62 oocytes, in each respective group, were suitable for analysis. The haploidy, diploidy and aneuploidy rates between ICSI (62.7, 7.8 and 5.9%) and IVF (61.3, 9.7 and 14.5%) groups were similar. A significant inter-patient variation in the incidence of hypohaploidy was observed within the IVF group. Chromosomal fragmentation or breakage was observed at a similar rate in both groups of unfertilized oocytes (23.5 and 14.5% for ICSI and IVF respectively). A significantly higher proportion of ICSI oocytes contained sperm nuclei (27/51, 52.9%) than did IVF oocytes (20/62, 32.3%, P < 0.01). The distribution and state of sperm head chromatin in relation to oocyte chromosomal complement was studied in both groups. ICSI oocytes contained decondensed or swollen sperm nuclei in association with haploid oocyte chromosomes (12/27, 44.4%) or condensed sperm heads in oocytes showing no chromosomal complements (7/27, 25.9%). In IVF oocytes sperm heads were either arrested in the condensed state (5/20, 25%), metaphase stage (3/20, 15%) or had undergone premature chromosome condensation (PCC; 6/20, 30%) in association with haploid oocyte chromosomes. The incidence of PCC was similar in the two groups. A marked variation in the incidence of total chromosomal abnormality was observed between patients within both ICSI (0-75%) and IVF (0-71%) groups indicating a possible similarity in oocyte quality between the majority of male factor and tubal infertility patients. The type of sperm used in the two fertilization procedures showed an increased incidence of chromosomal breakage with ICSI-MESA (microepididymal sperm aspiration) spermatozoa (4/6, 67%) compared to the ICSI-ejaculated (6/35, 17.1%; P < 0.05), ICSI-testicular biopsy (2/10, 20%) and IVF-normospermic (9/62, 14.5%; P < 0.01) spermatozoa. Chromosomal fragmentation may be associated with the degree of difficulty experienced at sperm injection, especially with sperm retrieved from the reproductive tract. Thus chromosomal fragmentation in ICSI may need further investigation using a larger sample size in order to assess the possible causative factors.     

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.     

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.     

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.     

In vitro fertilization treatment for severe male factor: a comparative study of intracytoplasmic sperm injection with testicular sperm extraction and with spermatozoa from ejaculate

PURPOSE: Our purpose was to evaluate whether the source of spermatozoa influences the results of intracytoplasmic sperm injection (ICSI) treatment in couples with severe male-factor infertility. METHODS: A retrospective analysis of 40 cases of ICSI with testicular-retrieved spermatozoa, matched with 40 cases of ICSI with ejaculated spermatozoa, was performed. We included only couples with normoovulatory females younger than 37 years who were matched according to the day of ovum pickup with the patients in the study group. RESULTS: Eighty cycles were analyzed: 40 cycles using testicular spermatozoa and 40 cycles using ejaculated spermatozoa. In 32 (80%) of the 40 ICSI transcutaneous needle aspiration cycles, we obtained enough spermatozoa to inject all the mature oocytes retrieved. In eight (20%) cases there were not enough spermatozoa to inject all the oocytes. Only 76 (54%) of 141 available oocytes were injected in these eight patients. The oocyte fertilization rates were 42% for the study group and 55.5% for the controls (P < 0.005). Thirty-six (90%) patients in the group with nonobstructive a zoospermia (NOA) and 37 (92.5%) patients in the oligoteratoasthenospermia (OTA) group had embryos for replacement. The mean cleavage rates per cycle (96% with testicular and 93% with ejaculated spermatozoa), the mean number of embryos per transfer (3.72 +/- 1.6 in the NOA group and 4.24 +/- 1.5 in the OTA group), the embryo quality (cumulative embryo scoring = 34.03 +/- 22.62 in the testicular sperm group and 36.08 +/- 19.28 in the ejaculated sperm group), and the clinical pregnancy rates (22.5% in the NOA patients and 20% in the ejaculate group) were not significantly different between groups. CONCLUSIONS: High fertilization, cleavage, and pregnancy rates can be achieved with intracytoplasmic testicular sperm injection from patients with NOA, reaching levels comparable with those of ICSI using ejaculated spermatozoa.     

A simple method for recovering the motile spermatozoa from extremely low quality sperm samples

Recovery of motile spermatozoa from extremely low quality samples for use in the intracytoplasmic sperm injection (ICSI) procedure is difficult. To solve this problem we developed a simple method to recover the motile spermatozoa using a 3% polyvinylpyrrolidone (PVP) droplet. After depositing a sperm pellet into this slightly viscous droplet, motile spermatozoa readily swam out to the clear area while immotile spermatozoa dispersed to a lesser extent, so that motile and immotile cells became clearly separated from each other. A total of 36 ICSI cycles using spermatozoa with extremely low quality characteristics were performed. We recovered the motile spermatozoa from all sperm samples from two sources of poor quality spermatozoa. Thirty-one cycles of ICSI with ejaculate resulted in fertilization and pregnancy rates of 54 and 29% respectively. Five cycles of ICSI with frozen-thawed epididymal spermatozoa resulted in fertilization and pregnancy rates of 70 and 60% respectively. The 3% PVP droplet method is very simple and easy to perform, so it may be useful for recovering the motile spermatozoa from extremely low quality sperm samples used for ICSI.     

Assisted fertilization by subzonal insemination and intracytoplasmic sperm injection

The results of 600 consecutive treatment cycles of subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) are described in couples with failed fertilization after standard IVF or insufficient spermatozoa in the ejaculate for IVF. More oocytes were damaged by ICSI (16.3%) than by SUZI (8.5%) and the normal fertilization rate was substantially higher after ICSI (49.1% v. 16.6%). Subsequent development of two-pronuclear oocytes in vitro was 80% after SUZI and 73.9% after ICSI. Significantly more triple embryo replacements were carried out after ICSI than after SUZI. Embryo transfers were possible in 421 of the 600 cycles. There were 63 pregnancies after ICSI (215 transfers) and 23 after SUZI (156 transfers); 10 additional pregnancies were achieved after 50 transfers of a mixture of SUZI and ICSI embryos. The results of fetal karyotypes and follow-up of the children do not indicate an increase in congenital malformations.     

Azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection

Some men with non-obstructive azoospermia harbour fully formed spermatozoa within their testicular tissue that can be used to achieve pregnancy via intracytoplasmic sperm injection (ICSI). Recently, Reijo et al. (1995) provided compelling evidence that the DAZ gene cluster is a strong candidate for one of the elusive azoospermia factors (AZF) located on the long arm of the Y chromosome. The DAZ gene cluster is deleted in 13% of azoospermic men and a small percentage of severely oligozoospermic men. Vertical transmission from father to son of AZF region deletions has also been described. Presumably these fathers were oligozoospermic. This led us to ask whether the azoospermic male with deletions of the AZF/DAZ region can also complete minimal spermatogenesis and whether any spermatozoa found could participate in fertilization, embryo development and pregnancy. Three out of six (50%) of the azoospermic men with AZF/DAZ deletions had spermatozoa identified within their harvested testicular tissue. When these spermatozoa were used for ICSI, fertilization occurred in 36% of injected oocytes. This compared favourably with testicular spermatozoa retrieved from non-obstructive azoospermic men without AZF/DAZ gene deletions. In one case, a twin conception resulted, which represents the first term pregnancy reported using spermatozoa from an AZF/DAZ deleted azoospermic male. Therefore it is necessary to take the possibility of transmission of infertility or sterility to our patients' offspring seriously when utilizing today's reproductive technologies, as spermatogenesis in men with AZF/DAZ deletions is by no means an exceptional occurrence.     

Investigation of epididymal sperm maturation in the golden hamster

During passage of hamster spermatozoa through the epididymis their maturation is shown to involve changes in the sperm head, midpiece (mitochondria) and tail. The sum of these changes results in a dramatic increase in the fertilizing potential of the spermatozoa. When comparable numbers of spermatozoa from the caput or corpus epididymis were injected into one uterine horn of mature females, following ovulation induction, and spermatozoa from the cauda epididymis were injected into the contralateral horn, no fertilization was observed with caput epididymal spermatozoa, 1.7% of oocytes were fertilized by corpus epididymal spermatozoa, whereas 79.5% fertilization was obtained with cauda epididymal spermatozoa. Total sperm numbers increased from caput to corpus to cauda [28.3 +/- 12.2, 40.6 +/- 20.8, 144 [corrected] +/- 62 million, respectively]. The percentage of progressively motile spermatozoa increased from 27.9 +/- 6.4 to 33.8 +/- 4.8 to 70 +/- 10.7 during this passage. Viability, measured by exclusion of the dye, propidium iodide, was significantly less in spermatozoa from the cauda than from the proximal or mid-caput epididymis. The percentage of the live cells that were stained intensely by rhodamine-123 (a measure of mitochondrial membrane potential) increased during epididymal passage from 22.8 +/- 7.8% in the proximal caput epididymis to 57.2 +/- 16.5% in the cauda epididymis. Staining with acridine orange (a measure of DNA packaging in the sperm head) indicated an increase in chromatin condensation in cauda epididymal spermatozoa, when compared to those obtained from the caput or corpus.     

The heparin-glutathione test: an alternative to the hypo-osmotic swelling test to select viable sperm for intracytoplasmic sperm injection

OBJECTIVE: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI). DESIGN: A prospective study. SETTING: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad. PATIENT(S): Semen samples from healthy donors and patients with infertility. INTERVENTION(S): Sperm samples were kept in culture for different periods in Ham's F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH-treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed. MAIN OUTCOME MEASURE(S): Sperm nuclear decondensation, progressive and nonprogressive motility, and male pronucleus formation. RESULT(S): The maximum proportion of sperm nuclear decondensation (28.7%+/-2.1% versus 2.6%+/-0.5% in the control group) was reached after 60 minutes of incubation in the presence of a heparin-GSH mixture. Differences in the percentages of progressive and nonprogressive motility among treatments and times of incubation, although statistically significant, were biologically negligible. No statistically significant differences were observed in the rate of sperm head decondensation (8.2% [4/49] versus 11.1% [6/54]) and male pronucleus formation (18.4% [9/49] versus 22.2% [12/541) after the injection of control and treated spermatozoa into hamster oocytes. CONCLUSION(S): The HEGLUT may offer an alternative to the hypo-osmotic swelling test for the selection of viable sperm for ICSI.     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.