Usefulness of partial dissection of the zona pellucida in a human in-vitro fertilization programme.

The influence of partial zona dissection (PZD) on the fertilization rate was studied in 34 couples with a history of fertilization failure and/or severe sperm deficiency. Overall, PZD improved the rate of monospermic fertilization compared to controls (41/254 versus 6/111: P less than 0.001) and fertilization was achieved in 50% of cases. However, the results differed according to the seminal characteristics. In 10 couples with at least two in-vitro fertilization (IVF) trials entailing total fertilization failure and with semen defined as normal, PZD did not significantly improve the monospermic fertilization rate (6/44 in the PZD group versus 2/39 among controls). A benefit related to PZD was evident in 33 attempts with severe sperm deficiency. In this group, only four of 72 unmanipulated control oocytes were fertilized but the monospermic fertilization rate was 14.6% for PZD oocytes. The rates of polyspermy were 40% and 14.6% in the groups with normal and abnormal semen parameters respectively. Of 33 trials with defective spermatozoa, 20 reached the stage of embryo transfer and three pregnancies were obtained, of which one aborted at 9 weeks.     

Routine application of partial zona dissection for male factor infertility

Partial zona dissection (PZD) increases the chances of fertilization by improving the access of spermatozoa to the perivitelline space (PVS) helping those spermatozoa unable to penetrate the zona pellucida (ZP) and possibly those poorly able to penetrate the oolemma. Problems arise in assessing semen to decide which parameters might indicate defects of this nature. PZD, by circumventing the ZP, may also increase the rate of polyspermy, especially in infertility where ZP and oolemmal penetration are not defective. Given these drawbacks, we performed PZD as routine treatment for male infertility in 70 in-vitro fertilization cycles. In three different groups, PZD proved to be either effective, ineffective or unnecessary. In the first group of 35 cycles, fertilization was 23% with initial PZD and 33% with PZD reinsemination (36% and 41% polyspermy respectively). No fertilization occurred following conventional insemination (CONV). Four pregnancies occurred in this group. In a second group of 19 cycles, fertilization did not occur with either PZD or CONV. In the final group of 16 cycles, fertilization was similar following both PZD and CONV, but polyspermy was 48% in the PZD category. Transfer of mixed PZD and CONV embryos in this group yielded 10 pregnancies. Assessment of all patient and seminal profiles, and those in an oligozoospermic subcategory, revealed no parameters of relevance to success or failure with PZD. However, one subgroup in the group of total failure to fertilize did have a significantly lower percentage of normal morphology (P less than 0.005), suggesting that degree of teratozoospermia may be a prognosticator of success using PZD.     

Microsurgical fertilization and teratozoospermia

Semen parameters were correlated with the outcome of partial zona dissection (PZD) in 42 couples with male factor infertility. Although fertilization rates were reduced, 12% of the embryos implanted following replacement. Spermatozoa from teratozoospermic sperm populations were able to fuse with oocytes following zona penetration through the artificial gaps. PZD followed by insemination with less than 5% normal spermatozoa led to 20 embryos which, upon replacement, did not implant. Motility and sperm count were not clearly correlated with the outcome of PZD and are therefore less useful indicators for patient selection. Teratozoospermic patients who previously failed to fertilize were compared to a group of similar patients who had not attempted IVF before. Although fertilization was significantly improved in first-time patients, 41% of the patients whose spermatozoa were initially unable to fertilize had at least one embryo when PZD was performed. Several pregnancies were established in this group. Subzonal sperm insertion (SZI) and PZD were compared in 19 patients using sibling oocytes. A significant fraction of spermatozoa from infertile men were able to fuse with the oolemma when directly inserted into the perivitelline area. Using a sucrose solution to shrink the ooplasm, only 1% of the oocytes were damaged during SZI. Monospermic fertilization rates following PZD and SZI were 15 and 16%, respectively. Both micromanipulation methods were successful in most patients. However, in two small groups of patients, only one technique resulted in fertilization.     

IVF treatment of moderate male factor infertility: a comparison of mini-Percoll, partial zona dissection and sub-zonal sperm insertion techniques

In this study we examined various techniques of in-vitro fertilization(IVF) for treating couples in whom the male had subnormal semenparameters. We compared two sperm preparation methods (mini-Percolland conventional swim-up) for efficiency of recovery after preparationand for fertilization rates after IVF, and compared the suitabilityof partial zona dissection (PZD) and sub-zonal sperm insertion(SUZI) to patients with different types of male factor infertility.The mini-Percoll technique allowed the recovery of significantlymore motile spermatozoa from the same semen sample comparedto the swim-up method. More oocytes were fertilized after spermatozoawere prepared by the mini-Percoll technique. An increased numberof spermatozoa recovered from an ejaculate led to an improvementin the quality of spermatozoa in the insemination droplet. Subsequently,when using the PZD technique, the fertilization rate increasedwhen there was a higher number of spermatozoa in the patient'sejaculate. When comparing the two micromanipulation techniques,SUZI provided patients with oligoasthenzoo-spermia (i.e. <10 x 10⁶ spermatozoa/ml and 40% motility) with a higher chanceof obtaining 2-pronculeate eggs.     

Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro

Zona drilling (ZD) and zona cutting (ZC) were used in an IVF programme to assist fertilization in semen defect patients. Twenty-seven patients consented to ZD where acidified Tyrode's was used to create a hole in the zona pellucida. In 19 patients, ZD increased the fertilization rate to 29% compared with 8% (P less than 0.001) in their routine IVF cycles, and in eight patients precluded from routine IVF, a fertilization rate of 14% was achieved. Twenty-two patients consented to ZC where a slit in the zona is made mechanically. In 12 patients ZC increased the fertilization rate to 31% compared with 14% (P less than 0.01) from previous routine IVF cycles, and in 10 patients precluded from routine IVF, a fertilization rate of 34% was achieved. In 13 cycles, 68 uncut control oocytes were inseminated. In five cycles both control and ZC oocytes were fertilized (n.s.d.). In eight cycles no control oocytes were fertilized compared with 27% of ZC oocytes. The polyspermy rate was 4.6%. Twenty-four per cent of ZD and 12% of ZC (P less than 0.01) oocytes and embryos were degenerate after 42 h. Both ZD and ZC can increase the fertilization rate of sub-optimal semen, however, in our hands neither technique produced a pregnancy.     

Subzonal insemination, partial zona dissection or intracytoplasmic sperm injection? An easy decision?

This review aims to analyse and compare the results to dateof subzonal insemination (SUZI), partial zona dissection (PZD)and intracytoplasmic sperm injection (ICSI) to evaluate criticallywhether it is now possible to replace SUZI and PZD by ICSI.It appears that ICSI is a much more efficient assisted reproductiontechnique than SUZI and PZD for resolving cases of severe maleinfertility and/or repeated failure of conventional in-vitrofertilization (IVF). For ICSI compared with SUZI and PZD, fertilization(49.4, 17.7 and 16.8% respectively), percentage of patientsreaching embryo transfer (91.0, 55.1 and 23.3% respectively),percentage of transfers performed with two or three embryos(83.3% ICSI and 39.3% SUZI), pregnancy rate per embryo replacement(28.2, 18.7 and 16.5% respectively) and pregnancy rate per oocyteretrieval (24.8, 10.3 and 3.8% respectively) are all improved.In addition, cases of severely impaired semen characteristics,which were condemned to infertility for life with conventionalIVF, SUZI or PZD, can now be treated and resolved efficientlywith ICSI.     

Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis.

Assisted hatching by zona drilling using acidic Tyrode's solution was performed during three randomized trials in 330 in-vitro fertilization patients. The trials were designed in order to study the overall effect of the procedure and whether characteristic patient [i.e. maternal age and basal levels of follicle stimulating hormone (FSH)] and embryonic features (i.e. zona pellucida thickness) are important for the decision to perform assisted hatching routinely. Couples (n = 137; Trial 1) in whom the female partner had normal basal FSH levels were randomized in a control group (without micromanipulation) and a zona drilling group (all embryos micromanipulated). The incidence of implantation (67/239; 28%) of zona-drilled embryos compared favourably with that of control embryos (49/229; 21%), but the difference was not significant. Retrospective analysis revealed that those embryos whose zonae were thicker than 15 microns were rescued. In order to test the validity of this finding, selective assisted hatching was performed on embryos with a poor prognosis in 163 other patients (Trial 2). The couples were randomized into a control group and a group in which embryos were selectively zona-drilled, based on zona thickness and other embryonic features. The rate of embryonic implantation in the selectively zona-drilled group was 25% (70/278), significantly (P less than 0.05) higher than that of control embryos (51/285; 18%). Although it was demonstrated retrospectively and prospectively that assisted hatching by zona drilling is effective in embryos with thick zonae (greater than or equal to 15 microns), patients whose embryos have thin (less than 13 microns) zonae may be jeopardized by the procedure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Andrology: Seventeen live births after the use of an erbium-yytrium aluminium garnet laser in the treatment of male factor infertility

Mechanical partial zonal dissection (PZD) is one of the micro-inseminationtechniques developed to improve the chances of fertilizationby in-vitro insemination in subfertile males. We developed anew and safe laser method (erbium laser ablation) for PZD, withthe aim of producing a precise opening as well as ablation ofsome layers of the zona pellucida. From February 1992 to March1993, 104 couples suffering from male factor infertility weretreated in our centre. All patients were affected by severeoligoasthenozoospermia and had previously undergone one failedin–vitro fertilization attempt each. Photoablation ofthe zona pellucida was induced in 512 oocytes by exposure toan erbium-yytrium aluminium garnet (Yag) laser. The openingsobtained were 14µm in diameter. Of the laser–treatedoocytes, 158 (30%) fertilized and 139 (88%) cleaved. Nineteen(18.3%) clinical pregnancies resulted and produced 17 newborns,all in good health. In our series there were four miscarriages,23 damaged oocytes (4.4%) and 13 with three or more pronuclei(2.5%). Considering that the incidence of damaged oocytes andpolyspermy is low, it seems that in some cases of male factorinfertility erbium—Yag laser photoablation of the oocytezona pellucida can be considered a procedure which is effectivein enhancing fertilization and which is safe, allowing normalembryo development.     

Opening of the mouse zona pellucida by laser without a micromanipulator

A contact-free laser system is described for ablation of theembryonic mouse zona pellucida using a pulsed excimer 308 nmlaser. Effects on further embryonic development were evaluated.Zonae of 8- to 16-cell mouse embryos were either lased (n =189), zona-drilled with acidified Tyrode's solution (n = 183)or left zona-intact (n = 188). Blastocyst formation (99–100%)was similar in the three groups. Hatching occurred earlier inlased embryos compared to those of the control group. Theseblastocysts hatched through the laser ablated area. Significantlymore embryos were hatching on day 4 in the conventionally drilledgroup when compared to the laser treated group (50% versus 24%respectively). On day 7 of development, significantly (P <0.05) more embryos conventionally zona-drilled (37%) were intactthan those which were previously laser treated (10%). Abnormaldevelopment was also noted in a small group of embryos whichwere lased just on the outside of the zona in comparison to1/3 of an embryonic width away from the zona. The current resultssuggest that apparently precise zona laser ablation with anexcimer laser at 308 nm may have potential adverse effects whichmay only be manifested after a prolonged period of culture pastthe cavitation stage. However, implantation rates of morphologicallynormal laser abalated embryos were not impaired when comparedto control embryos.     

Improved hatching in mouse embryos brought about by combined partial zona dissection and co-culture

In this study 874 mouse embryos were allocated to six groupsincluding a control, co-culture, and four groups that underwentpartial zona dissection (PZD): at the 2-cell (PZD-2) and morulastages (PZD-M) both with and without co-culture. Rates of completeblastocyst hatching on day 5 increased in the following order:control, co-culture alone, PZD-2 alone, PZD-M alone, PZD-2 withco-culture and PZD-M with co-culture (P < 0.00001). PZD-Mled to significantly higher rates of complete blastocyst hatchingcompared to PZD-2 (P < 0.03). This study showed also thatco-culture apparently compensates for any minor damage incurredduring the PZD technique at the 2-cell and morula stages, (P< 0.01 and P < 0.01) respectively. Therefore PZD and co-cultureseem mutually beneficial techniques that promote early blastocysthatching in the mouse.     

Zona opening with 308 nm XeCl excimer laser improves fertilization by spermatozoa from long-term vasectomized mice

Spermatozoa from long-term vasectomized mice have greatly reducedfertilizing ability in vivo and in vitro, which makes this auseful animal model for male factor infertility. The purposeof this study was to evaluate the 308 nm XeCl excimer laserfor opening the zona pellucida to enhance the fertilizationrate with spermatozoa from vasectomized males. Inseminatingzona-intact (control) oocytes with 5 x 10⁶ spermatozoa/ml resultedin only 6% fertilization and 33.3% development to the blastocyststage; zona-opened oocytes showed significant improvement with31.5% fertilization, 90% cleavage to the 2-cell stage, and 72.2%blastocyst formation. Out of the 130 oocytes in the experimentalgroup, zona ablation was performed successfully on 127 and onlythree were damaged. These results suggest that laser micromanipulationfor assisted fertilization potentially offers a simplified andprecise method for mechanical zona cutting.     

Fertilization and early embryology: Ascorbate-supplemented media in short-term cultures of human embryos

The present study aimed to evaluate whether ascorbate, a reactiveoxygen species (ROS) scavenger, can improve fertilization anddevelopment of human embryos in vitro when added to the simplesalt solution human tubal fluid (HTF) or the complex tissueculture medium Ham's F-10, which contains iron and copper inits formulation. Human oocytes, spermatozoa and embryos from83 infertile IVF couples were randomly allocated and culturedin the presence or absence of 62.5 _µM ascorbate in HTFmedium (39 couples) or Ham’s F-10 medium (44 couples).No significant effect of ascorbate on fertilization, numberof cells and embryo grade per embryo on days 2 and 3 after insemination,or percentage of embryos showing developmental block on day3 (those embryos that were still at the 2-cell stage) was observedwhen data were analysed together or divided into several groupsaccording to the cause of infertility, quality of semen sampleused for insemination and women‘s age in either of thetwo media tested. Despite these results, a positive effect ofascorbate on fertilization and embryo development in vitro cannotbe totally ruled out until the effects of other, non-physiologicalconcentrations of ascorbate and longer-term embryo cultures(to the blastocyst stage) have been tested.     

Fractured zona oocytes in in-vitro fertilization cycles stimulated with gonadotrophin-releasing hormone analogue and human menopausal gonadotrophin

In order to assess the possible influence of gonadotrophinreleasinghormone analogue and human menopausal gonadotrophin on the occurrenceof fractured zona oocytes (FZOs) in in-vitro fertilization (IVF)treatment cycles, we analysed 267 consecutive cycles in 199patients. In 87 cycles, at least one fractured zona oocyte wasrecovered, and in 180 cycles only intact zona oocytes (IZOs)were recovered. FZOs represented 5.8% of all oocytes retrievedand 14.8% when only cycles with FZOs were considered. Serumoestradiol concentrations were significantly higher at day –3and day –2 (P < 0.02) in cycles yielding at least onefractured zona oocyte compared to IZO cycles (day 0 = retrievalday), and there was a higher incidence of G terminal patternof oestradiol curve (P < 0.01) in cycles with FZOs. The meannumbers of all oocytes retrieved and of mature oocytes weresignificantly higher in FZO than in IZO cycles (P < 0.001).The fertilization rate of mature oocytes was significantly reduced(P < 0.05) in cycles with one or more oocytes with fracturedzonae. There was no significant difference in the number ofembryos transferred, pregnancy and abortion rates in both groups.We conclude that although the occurrence of fractured zona oocytesis a frequent event, it does not affect the overall resultsof our IVF programme. Zona pellucida fragility may be the resultof over-maturation of some oocytes.     

Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 {micro}m diode laser beam

The safety of microdrilling the zona pellucida of moose oocyteswith a 1.48 µm diode laser has been investigated by determiningthe ability of mouse oocytes to fertilize in vitro and developin vivo. Mice born after transfer of control and zona pelludda-microdrilledembryos into foster mothers were submitted to anatomical andimmunohisto-chemical investigations, and their aptitude to breedwas assessed in two subsequent generations. Decolonization ofthe oocytes with hyaluronidase induced a reduction of the fertilizationand implantation rates, which was attributed to a zona hardeningphenomenon. After laser zona pellucida microdrilling, theserates were restored to those obtained with embryos derived fromuntreated oocyte-cumulus complexes. Pups derived from zona pellucidamicrodrilled embryos were comparable with those obtained fromcontrol embryos, confirming the lack of deleterious effectsof the laser treatment In conclusion, the 1.48 µm diodelaser allows safe microdrilling of the zona pellucida of mouseoocytes after decoronization with hyaluronidase. Based on thehealth of the F2 generation and the lack of neuroanatom-icaland neurochemical differences, we concluded that this technologymay be investigated in the human, particularly when the zonapellucida represents the main impediment for fertilization orembryo hatching.     

Fertilization rate in couples with unexplained infertility.

A group of 24 couples with unexplained infertility was scheduled for in-vitro fertilization and tubal embryo transfer between May 1989 and September 1990. In the same period, in-vitro fertilization and intrauterine transfer of embryos was planned in a control group of 44 women with tubal infertility. The mean age and duration of infertility were similar in both groups and the same scheme of ovarian stimulation was used. No statistically significant difference was obtained comparing oestradiol levels and numbers of mature oocytes retrieved between the group of patients with unexplained infertility and those with tubal infertility. The fertilization rate of the oocytes obtained from women with unexplained infertility (60.4%) was significantly lower (P less than 0.001) than that of the oocytes obtained from patients with tubal infertility (87.3%). There was no statistically significant difference in the cleavage rates between patients with unexplained infertility and those with tubal infertility. It is concluded that lack of fertilization is an unexplored cause of infertility in couples with unexplained infertility.     

Fertilization and early embryology: Evidence of sperm entry into assumed unfertilized human oocytes after sub-zonal sperm microinjection

Sub-zonal sperm microinjection (SUZI) as a treatment for malefactor infertility can facilitate fertilization, however, inmany cases oocytes remain unfertilized even though the spermis placed in close contact with the oolemma. In order to improveour understanding of gamete interaction in cases of failed fertilization,we have analysed the failed fertilized oocytes from both SUZIand conventional in-vitro fertilization. The fluorochrome Hoechst33342 (which binds specifically to DNA) was used to check forthe possible presence of paternal chromatin in the unfertilizedoocytes. A significantly higher (P < 0.01) number of microinjectedoocytes showed signs of fertilization 2–3 days after spermmicroinjection compared to normally inseminated oocytes, 30/175(17.1%) and 2/79 (2.5%) respectively. In addition, four outof eight couples returning for a second treatment by SUZI displayedanomalies in fertilization in both cycles. The semen characteristicsof patients with or without anomalies in fertilization was notdifferent. The irregularities observed in the fertilizationprocess infer that certain male factor patients have intrinsicsperm anomalies lying at the sperm membrane and/or chromatinlevel that could lead to anomalies in the appearance of thepronuclei.     

Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen.

An auto-controlled study was conducted in couples with tubal infertility and normozoospermic semen. The fertilization rates and embryonic development in sibling oocytes treated, using the same semen sample, either by conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at the same time were compared. Sibling oocyte-cumulus complexes (OCC) of 56 different couples with tubal infertility and normozoospermic semen were randomly divided in order of retrieval into two groups inseminated either by conventional IVF or by ICSI. Of the retrieved OCC in the same cohort, 53.0 +/- 31.2 and 62.0 +/- 26.6% showed two distinct pronuclei after conventional IVF and ICSI respectively (not significant). Complete fertilization failure occurred after conventional IVF in 12.5% (7/56 couples). After ICSI, the comparable figure was 3.6% (2/56). The number of cases was too small to apply a statistical test to this difference. Total cleavage rates were quite similar: 86.7 +/- 28.0 and 90.1 +/- 21% of the zygotes developed into transferable embryos after IVF and ICSI respectively (not significant). Similarly, no difference in embryo quality was observed. Although injection and insemination of the oocytes were performed at the same time in the two groups, at 42 h post-insemination more embryos were at the four-cell stage after ICSI (P < 0.001) than after conventional IVF, where more embryos were still at the two-cell stage (P < 0.02). Embryo transfer was possible in all 56 couples, resulting in 16 positive serum human chorionic gonadotrophin tests (28.6% per embryo transfer), from which a clinical pregnancy resulted in 15 couples. The best embryos were selected for transfer independently of the insemination procedure, but preferably from the same origin. There appeared to be no difference in implantation potency of the embryos obtained with either technique after the non-randomized transfers.     

The impact of cigarette smoking on zona pellucida thickness of oocytes and embryos prior to transfer into the uterine cavity

BACKGROUND: Smoking has been reported to promote infertility. The zona pellucida plays an important role in fertilization and implantation. We report, for the first time, the effect of cigarette smoking on zona pellucida thickness of oocytes and embryos as one of the factors that may interfere with fertility. METHODS: This study comprised 169 women, grouped according to their smoking habits: 31 active smokers, whose husbands do not smoke; 44 active smokers, whose husbands smoke; 65 passive smokers, because of smoking husbands and 29 non-smokers (women and husbands). Zona pellucida thickness was measured prospectively on printed photos of 903 oocytes and 456 embryos. RESULTS: The zona pellucida thickness of oocytes and embryos of non-smoking women was significantly thinner than those of active and passive smokers. However, no significant differences were observed in the natural ability of the zona pellucida to become thinner after 48 h in culture. CONCLUSIONS: Our study demonstrates that active and passive cigarette smoking increases the zona pellucida thickness of oocytes and embryos. Our findings also show that active and passive smoking has no significant effect on the thinning mechanism of the zona pellucida, which implies that it is independent of the initial zona pellucida thickness.     

Fertilization in vitro of human oocytes by spermatozoa collected in different stressful situations

It has been shown that semen quality is impaired in couplesundergoing in-vitro fertilization (IVF), probably due to stress.A possible effect of stress on the ability of spermatozoa tofertilize human oocytes in vitro was analysed in the presentstudy composed of 26 couples with normozoospermic men undergoingIVF. A semen sample was obtained during the infertility work-upand was cryopreserved (sample 1). A second sample (sample 2)was provided after oocyte retrieval during the IVF cycle. Sample1 was thawed and both samples were washed and preincubated foroocyte insemination. One-hundred-and-five oocytes were inseminatedusing thawed sample 1, and 120 with sample 2.Semen parameterssuch as density, progressive motility and percentage of abnormalforms were compared between sample 1, before and after freezing,and sample 2. Only motility was significantly (P<0.01) decreasedby cryopreservation in sample 1, but no parameter was significantlydifferent when fresh sample 1 was compared to sample 2. Thefertilization rate was 78.6% using sample 1 in comparison to87.5% when sample 2 was employed (not significant, NS). Cleavagerates were 77.7 and 89.7%, respectively (NS). A group of fivepatients undergoing IVF who needed donor semen served as a controlfor the effect of sperm cryopreservation on IVF. In these cases,the donor was asked to provide a fresh sample. Half of thissample was frozen and thawed. Subsequently, fresh and thawedsamples were prepared for insemination and oocytes inseminatedeither with the fresh preparation (n=24) or the frozen and thawedspermatozoa (n=22). There was a significant (P<0.05) decreasein motility in the thawed sample, but fertilization and cleavagerates were not different. These data suggest that the stressfulsituation induced by IVF treatment in normozoospermic men doesnot affect the ability of spermatozoa to fertilize human oocytesin vitro. Cryopreservation of human spermatozoa before IVF maybe a good policy in couples especially suspected of being understress during this procedure.