Partial Zona Dissection Research Articles

Improvement of fertilizing performance by normal and abnormal mouse semen after zona opening of mature oocytes

The usefulness of opening the zona pellucida by partial zona dissection (PZD) to enhance fertilization of mature mouse oocytes was studied after insemination with three types of semen: normal and diluted semen and semen from long-term-vasectomized males. Zona opening did not by itself induce parthenogenetic cleavage of mature oocytes and did not significantly increase mono- and polyspermic fertilization of oocytes inseminated with normal semen. While a fertilization rate of 62% was obtained among intact oocytes, of which 4.5% were polyspermic, a 66.8% fertilization rate was observed among PZD oocytes, 6.3% of which were polyspermic. However, after using diluted semen, only 54 of 193 intact oocytes were fertilized (28%), and PZD improved the fertilization rate to 65.4%. Cleavage rate of nonmanipulated oocytes inseminated with abnormal semen from vasectomized males was dramatically decreased in comparison with those inseminated with normal semen (7.6% vs. 65%). PZD induced a moderate but significant improvement of fertilization performance when using this abnormal semen (19.6%).

Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology

To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. Two studies were performed: partial zona dissection and IVF was applied in 57 couples during the first period, and subzonal sperm insertion was also applied in a second group of 47 couples. Procedures were performed in an academic research environment. Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. Oocytes were micromanipulated with either partial zona dissection or subzonal sperm insertion, or the zona pellucida was left intact. Embryos were replaced in patients prophylactically treated with methylprednisolone and antibiotics. Because several microsurgical fertilization techniques are now available, this study was performed to compare sperm parameters, embryo morphology, fertilization, and implantation rates after application of two successful micromanipulation procedures. Twenty-one pregnancies were established in 104 patients, 5 definitely from subzonal sperm insertion and 4 from partial zona dissection. Patients who failed IVF before had a similar chance of pregnancy after the use of micromanipulation, as first time patients (9/53 versus 12/51). In a subgroup of 15 patients who failed IVF with insufficient numbers of motile sperm, fertilization was significantly higher after subzonal sperm insertion. Partially zona-dissected embryos from couples with severe teratozoospermia (less than or equal to 5% normal forms; strict criteria) had significantly more morphological abnormalities than those from patients with moderate teratozoospermia (6% to 10% normal forms). In severely teratozoospermic patients, significantly fewer partially zona-dissected than subzonally inserted embryos implanted. The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.

Assisted reproductive technology

Several aspects of reproductive technology are discussed. In tubal infertility, the choice between surgery or in vitro fertilization and embryo transfer (IVF-ET) is addressed. In cases with bilateral distal occlusion or otherwise bad prognosis, IVF is probably more successful and less expensive. IVF in unstimulated cycles has given promising results, with pregnancy rates comparable to the results from the National IVF-ET Registry. If these results can be confirmed by more studies they will probably have a great impact on the choice of treatment in tubal infertility. The results obtained with various transfer procedures in nontubal infertility have still to prove tubal transfer to be a more effective procedure than IVF. Finally, studies on microinsemination in male infertility or unexplained infertility with previous fertilization failure in IVF show promising results with the subzonal insemination and partial zona dissection procedures.

Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo

The present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure-eight shape. Seventy-two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P less than 0.001) more AT-blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT-embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD-embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.

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.

Micromanipulation in a center for reproductive medicine

Several variations of micromanipulation of the female gamete (zona drilling, zona cracking, ooplasmic sperm injection, partial zona dissection) have been applied recently to human IVF to overcome severe male factor. Of the first 16 cycles attempted using partial zona dissection, one pregnancy resulted leading to a normal term delivery. Careful removal of the coronal cells, as well as stepwise removal of sucrose postpartial zona dissection, will facilitate this procedure and greatly reduce potential damage to the oocyte by pH, mechanical, or thermal injury. Micromanipulation has become a routine service offered in our program in cases where the likelihood of a poor IVF outcome is either known or suspected, and also serves as a replacement for simple reinsemination in cases of failed fertilization.

Zona drilling enhances fertilization by mouse caput epididymal sperm

Spermatozoa from the caput epididymis are known to be much less capable of fertilization when compared to sperm from more distal segments of the epididymis. The purpose of this study was to determine if two micromanipulative techniques, zona drilling (ZD) and a modification of partial zona dissection (PZD), could be used to enhance fertilization with caput epididymal sperm. A mouse in vitro fertilization model was used. Inseminating oocytes with 500-1,000 sperm/oocyte from the cauda epididymis as a control resulted in fertilization of 98 of 300 (32.6%) oocytes. Of those fertilized, 47 developed to the blastocyst stage (47.9%). Caput sperm fertilized 13 of 116 (11.2%) nonmanipulated oocytes. Only 1 of 13 developed into a blastocyst, while with oocyte ZD, caput sperm fertilized 24 of 144 (16.7%) oocytes, 50% of those fertilized developing to blastocyst (P = 0.0129). When modified PZD was performed on oocytes, only one of 23 was fertilized, with no blastocyst development. These results indicate that acid Tyrode ZD enhances both fertilization and early embryonal development when caput epididymal sperm are used for insemination. These mouse studies suggest that ZD or other micromanipulation techniques may prove clinically useful in men with proximal epididymal obstruction where only caput sperm are available.

Clinical evaluation of three approaches to micromanipulation-assisted fertilization

Three different micromanipulation procedures were used to assist human fertilization in cases of severe male factor infertility. Zona drilling was performed either with acid Tyrode's solution, mechanically following zona softening with chymotrypsin, or by partial zona dissection. The fertilization rate was lowest in the zona drilling/acid Tyrode's group (7/40; 17.5%), although no differences between groups (zona drilling/chymotrypsin: 21/84, 25%; partial zona dissection: 31/143, 21.7%) were significant. The fertilization rate was significantly increased relative to untreated eggs from the same patients only in the partial zona dissection group (31/143, 21.7% versus 4/102, 3.9%). Oocyte damage occurred at a high rate as a result of zona drilling with acid Tyrode's solution (13/41, 37%). Embryonic development was compromised after zona drilling with chymotrypsin: only 7/12 (58.3%) of the fertilized oocytes cleaved, and the morphology of many of the cleaved embryos was abnormal. Although only 61% (16/26) of the diploid embryos resulting from partial zona dissection cleaved, the embryonic morphology of these embryos was comparable with controls. No pregnancies resulted from the transfer of manipulated embryos. We conclude that although zona manipulation increases the fertilization rate, losses due to oocyte trauma, low rates of diploid fertilization, low rates of cleavage, and a high frequency of abnormal cleavage reduce the number of embryos available for transfer.

Human sperm and hamster oocyte interaction: a model system to assess sperm entry into the oocyte after partial zona dissection

A model system of hamster oocyte and human sperm interaction was used to assess sperm entry into the perivitelline space after partial zona dissection. The procedure of zona dissection was standardized by creating slits which included one fourth, one eighth, one eighth X 2, or one sixteenth of the zonal circumference. Manipulated eggs were allowed to interact with 1 X 10(6) sperm/mL for 3 hours. A single large or medium slit was equally effective in permitting sperm entry into 46% and 47% of the manipulated eggs, respectively. However, the longer slit doubled the average number of sperm detected in the perivitelline space. A second medium-sized slit increased the rate of sperm entry into the perivitelline space to 76%, but the incidence of damaged oocytes also increased. A small slit did not permit sperm entry into any of the manipulated oocytes. This heterologous system of gamete interaction provides a model to evaluate requirements for successful partial zona dissection or other related procedures for assisted fertilization in the human.

Laboratory techniques for handling gametes and embryos

This review describes some basic and novel principles pertaining to the science of in vitro fertilization and embryo culture. Several systems for cell-free culture of gametes and embryos are described. The advantages of a coculture or helper cell system was investigated in several clinical trials using bovine cells from the reproductive tract. Improved embryonic morphology and implantation indicate that standard IVF culture provides suboptimal conditions. Microsurgical methods for assisting in vitro fertilization in cases of severe male factor infertility are also discussed. Thus far, it has been demonstrated that mechanical opening of the zona pellucida by methods such as partial zona dissection enhances fertilization. In addition, the introduction of a limited gap in the zona seems to facilitate hatching of the blastocyst, which may benefit all patients undergoing in vitro fertilization.

Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos.

Hatching in vitro was studied following zona drilling of 507 two-cell mouse embryos using three methods: 1) acidic Tyrode's (AT), 2) partial zona dissection (PZD) using a sharp microneedle, and 3) zona chiseling (CH), using a large beveled needle. PZD and CH were performed while the embryos were kept in a sucrose/PBS solution. Hatching was compared to 191 unmicromanipulated controls. The incidences of cavitation and completion of hatching did not differ between groups, however more micromanipulated embryos (20-25%) hatched partially than controls (9%). The zona pellucida thinned in 59/59 (100%) control blastocysts during expansion, but in only 3/205 (2%) micromanipulated blastocysts. The hatching gap was wide in all control embryos, but smaller in 96/129 (75%) micromanipulated embryos. Partially hatched blastocysts with a "figure-8" shape were found in 59/129 (46%) micromanipulated embryos and in none of the 39 hatching controls. Hatching usually occurred a day earlier in micromanipulated embryos as 214/218 (98%) had started extruding on day 5 as compared to 20/59 (27%) control blastocysts. Fifty percent of 1-day-old human oocytes were fertilized following PZD and reinsemination and 15/31 (48%) were monospermic. Thirteen monospermic embryos cleaved, six compacted and four cavitated--of these, three extruded through the PZD incision upon expansion. The zonae did not thin and one blastocyst twinned spontaneously as it was caught between the thick ridges of the PZD hole. Results indicate that the hatching process is abnormal following zona drilling; more embryos start hatching, extrusion occurs earlier, and many become trapped which may lead to artificial twinning or the formation of trophoblastic vesicles.

Monospermy and polyspermy after partial zona dissection of reinseminated human oocytes.

Partial zona dissection (PZD), a zona drilling method that uses mechanical force to open the zona pellucida while the oocyte is shrunken in a sucrose solution, was applied to 121 unfertilized 1-day-old mature human oocytes prior to reinsemination. The 115 surviving oocytes were divided into three groups in which the duration between sucrose addition and reinsemination was varied: I) Less than 20 minutes, II) 21 to 45 minutes, and III) longer than 45 minutes. There was a trend toward a reduced fertilization and polyspermy rate as the time between sucrose exposure and insemination in sucrose-free medium increased. Moreover, there was a statistically significant reduction in the number of oocytes penetrated by more than four sperm in group III (0/41) versus group I (7/34), and in group III, parthenogenetic development was observed. The incidence of polyspermy was also increased in oocytes manipulated more than 25 hours after retrieval compared with those manipulated 21-24 hours after recovery, supporting the idea that aged oocytes have a reduced ability to block polyspermy. Oocyte contraction in sucrose occurred in three different patterns: spherical, pear-shaped, and crenated. Both the fertilization and polyspermy rates were significantly higher in the crenated group. These results indicate that changes resembling activation occur following sucrose exposure and that sucrose activation can be used to reduce the risk of polyspermic fertilization in zona drilling procedures. In addition, the pattern of shrinkage in sucrose can be used as an indicator of oocyte receptivity to sperm penetration.

Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated.

Partial zona dissection (PZD) of human oocytes facilitates sperm penetration through mechanically made holes in the zona pellucida. Only 1 of 69 eggs was damaged when sucrose was used to shrink the ooplasm during micromanipulation. The fertilization rate of micromanipulated oocytes in 18 couples with male factor infertility was 68% (34/50), which compared favourably with insemination of non-micromanipulated controls (21/45, 47%). PZD was advantageous in oligozoospermic patients, but not in cases of asthenozoospermia, combined semen problems or immunological infertility. Three twin and two singleton pregnancies resulted following replacement of 23 micromanipulated and eight control embryos in 14 patients. No differences in embryo morphology and development rates were found between the micromanipulated and control groups. The incidence of polyspermy in couples with abnormal semen analyses was relatively low (less than 20%) possibly due to partial activation of the oocytes following exposure to sucrose. Polyspermy was high (57%) in normozoospermic patients with either immunological infertility (n = 3) or failure of fertilization in previous cycles (n = 4). In the three immunological patients, nine of 11 hyaluronidase and sucrose-exposed control embryos fertilized and six implanted, possibly indicating that cumulus and corona cells are contributing factors inhibiting fertilization in such cases.

Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration

Partial zona dissection (PZD), a method using mechanical force to open the human zona pellucida, and zona drilling, which uses acidic Tyrode's (AT) medium, were compared in 1-day-old oocytes prior to reinsemination. The incidences of monospermy and polyspermy were 13/54 (24%) and 14/54 (26%) following PZD and 6/46 (13%) and 8/46 (17%) following the use of AT medium. This compared favorably with conventional reinsemination: 15/161 (9%) monospermy and 4/161 (3%) polyspermy. Three of the 27 PZD embryos became blastocysts, while none of the AT-exposed embryos developed satisfactorily. Eleven male-factor couples had some of their oocytes randomly treated with PZD prior to insemination; each of the patients had non-micromanipulated control oocytes. Monospermic fertilization and cleavage (23/34; 68%) doubled (P less than 0.05) when PZD was compared with the control oocytes (10/30; 33%). Replacing two PZD and a single control embryo in two patients resulted in twin pregnancies. A third twin pregnancy was established following replacement of only micromanipulated embryos.