Beth E. Talansky

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

OBJECTIVE To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. DESIGN 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. SETTING Procedures were performed in an academic research environment. PATIENTS, PARTICIPANTS Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. INTERVENTIONS 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. MAIN OUTCOME MEASURES 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. RESULTS 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. CONCLUSIONS The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.

Controversies and opinions in clinical microsurgical fertilization

Gamete micromanipulation has enabled the reproductive biologist to bypass inefficient steps in the fertilization process. However, some methods have limited clinical benefit and their application has met with resistance due to ethical and legal restrictions. Though it is estimated that over 150 live births have resulted from microsurgical fertilization methods, most notably from partial zona dissection (PZD) and subzonal insertion of sperm (SZl, SUZl, or MIST), their general efficiency is being disputed. We have performed PZD in conjunction with SZl in 375 patient cycles without the use of lower semen analyses cutoff criteria. Though the clinical use of both methods appears promising, true success, measured as an increase in monospermic fertilization as well as enhanced implantation rates, has been reported by only a limited number of in vitro fertilization (IVF) programs (reviewed in Ref. 1). Basically there are four areas of controversy that surround the application of microsurgical fertilization procedures as clinically valid tools. (i) Does microsurgical fertilization truly improve the chance of having embryos for replacement (application of controlled studies)? and (ii) What patient selection criteria should be used? These two important issues are discussed separately below. (iii) While there is little doubt that PZD and SZl improve sperm fusion, excessive rates of polyspermy may lessen clinical efficiency (reviewed in Ref. 1). (iv) The implanting capacity of PZD and micromanipulated

Enhancement of Fertilization and Hatching Using Micromanipulation

Over 50 babies have now been born following the application of micromanipulation to oocytes and embryos. The current proceedings deal almost uniquely with the genetic diagnosis of biopsied polar bodies, blastomeres and nuclei from blastomeres or trophoblastic cells, however, micromanipulation procedures in assisted reproductive technology are performed for a variety of other reasons. Although the purpose of preimplantation diagnosis may differ from that of other microsurgical technologies, these methods have in common that the zona pellucida is breached. When normal fertilization is absent due to abnormalities in the sperm or oocytes, micromanipulation has been used to promote sperm-egg fusion (Malter and Cohen, 1989a; Ng et al, 1990). When Polyspermic fertilization occurs, micromanipulation may be used to return the zygote to a genetically normal, viable state (Gordon et al, 1989; Malter and Cohen, 1989b). Also, micromanipulation has been used to promote implantation by assisting the hatching process (Cohen et al, 1990a). However, micromanipulation imposes artificial conditions on the gametes and embryos.

An intact zona pellucida is not necessary for successful mouse embryo cryopreservation**Presented in part at the Sixth World Congress of In Vitro Fertilization, Jerusalum, Israel, April 2 to 7, 1989.††Supported in part by grant HD25136 from the National Institutes of Health, Bethesda, Maryland.

OBJECTIVE To determine the developmental potential of mouse embryos that underwent cryopreservation after micromanipulation of the zona pellucida. DESIGN Gaps were produced in the zona pellucida of mouse oocytes or two-cell embryos by zona drilling with acid Tyrodes solution. Zona-drilled oocytes were fertilized in vitro and cultured to the two-cell stage. Two-cell embryos were frozen, thawed, and cultured to the expanded blastocyst stage. RESULTS There was no difference in the rate of embryo survival post-thaw (248/318, 77% versus 288/345, 83.4%), or in the rate of development to the expanded blastocyst stage (91/248, 36.7% versus 88/288, 30.6%), between embryos that were zona drilled as oocytes and unmanipulated controls. Similarly, there was no difference in the rate of cryosurvival (206/217, 94.9% versus 168/187, 89.8%) or development to the blastocyst stage (154/206, 74.7% versus 132/168, 78.6%) between embryos that were fertilized in vivo and zona drilled at the two-cell stage and embryos that were unmanipulated. CONCLUSIONS These findings indicate that small gaps in the zona pellucida, such as those that result from micromanipulation, do not significantly alter the ability of embryos to withstand cryopreservation.

Techniques for Microsurgical Fertilization

During the past two years, pregnancies have been reported following the use of two microsurgical fertilization techniques: Partial Zona Dissection (PZD) and Subzonal Insertion (SI or MIST for microinsemination sperm transfer) (Cohen et al, 1988; Ng et al, 1988). To date, 25 pregnancies have been established with 12 healthy babies born from the application of PZD in 7 clinics worldwide. The pregnancy and birth rates from SI are not definite, although at least four pregnancies and one birth have been reported (Ng et al, 1990; Fishel et al, 1990).

Clinical evaluation of three approaches to micromanipulation-assisted fertilization**Presented in part at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November 13 to 16, 1989.

Three different micromanipulation procedures were used to assist human fertilization in cases of severe male factor infertility. Zona drilling was performed either with acid Tyrodes solution, mechanically following zona softening with chymotrypsin, or by partial zona dissection. The fertilization rate was lowest in the zona drilling/acid Tyrodes 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 Tyrodes 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.