Subzonal Sperm Insertion Research Articles

Gamete microsurgery for assisted fertilization.

Several methods of gamete microsurgery have been proposed to facilitate the union of the spermatozoon and oocyte in infertile couples. Partial zona dissection, subzonal sperm insertion, and intracytoplasmic sperm insertion are the three techniques currently being used for assisted fertilization in human in vitro fertilization laboratories. Fertilization and pregnancy rates after partial zona dissection and subzonal insertion have been variable and rather low. Recent reports of relatively high fertilization and pregnancy rates achieved after intracytoplasmic sperm insertion have rejuvenated interest in gamete microsurgery. Methods for each procedure are described. Studies which have attempted patient selection for gamete microsurgery are discussed.

Infertile couples with normal counts who require subzonal sperm insertion possess a fertility defect that affects zona pellucida penetration.

The results of subzonal sperm insertion (SUZI) have been retrospectively analysed in a subset of patients with normal sperm counts who were found to require SUZI because of poor or absent fertilization of zona-intact oocytes. This patient group is of particular interest because male factor-related infertility cannot be due to insufficient numbers of spermatozoa reaching the oocytes. Thus, failed fertilization can be attributed to deficiencies in one or more steps in the fertilization process, and SUZI provides a method of distinguishing defects of zona pellucida penetration from gamete fusion. A total of 26 such patients were treated identically to and concurrently with a much larger group of SUZI candidates who typically suffered from oligozoospermia, and fertilization results were compared. Fertilization rates after SUZI were higher in patients with normal counts than in oligozoospermic patients (51 and 26% respectively), indicating that the proportion of spermatozoa capable of fusing with the oocyte is the same or higher in the group with normal counts. In addition, nearly all SUZI procedures led to fertilization (23/26), with two out of three failed fertilizations occurring in cases where two or less oocytes were manipulated, results which further indicate that failed fertilization in these patients is not due to a defect at the level of gamete fusion. These findings suggest that infertility in these patients is based upon the inability of the spermatozoa to reach the oolemma and thus, that their fertility defect resides at the step of zona penetration.

Assisted reproduction and the male

Male reproductive problems are the commonest single cause of infertility and respond poorly to conventional treatment. Because of this, and because of the importance of co-existing female pathology, the techniques of assisted reproduction have acquired an important place in the management of male infertility. For many couples with a male factor, conventional in vitro fertilisation and embryo transfer will be appropriate. However, for those who fail with IVF, or whose semen is too poor (perhaps below 0.5 × 10 6 progressive spermatozoa/ejaculate), the techniques of micro-assisted fertilisation show much promise. These include partial zona dissection (PZD), sub-zonal sperm insertion (SUZI) and intracytoplasmic sperm injection (ICSI) the latter showing much promise, with pregnancy rates in excess of 30% per cycle being reported. However, the diagnosis of ‘male-factor’ infertility remains problematic, as does the intelligent selection of couples for treatment by these techniques.

Platelet activating factor treatment of spermatozoa enhances rabbit oocyte fertilization following subzonal sperm insertion.

The objective of this study was to evaluate the effects of spermatozoal platelet activating factor (PAF) treatment on fertilization rates employing subzonal sperm insertion in the rabbit. Oocytes (n = 130) with distinct first polar bodies were injected with three to seven motile spermatozoa. Half (n = 65) of the oocytes were injected with PAF-treated sperm, whereas the other half (n = 65) served as controls and were injected with nontreated sperm. Fertilization rates were assessed by the formation of two pronuclei followed by cleavage to the two-cell stage. In the group of oocytes injected with PAF-treated sperm, we noted a significant (P < 0.001) improvement in fertilization rates, 61.5% (40/65), vs. control nontreated sperm, 20.0% (13/65). Subsequent cleavage to the two-cell stage was noted for 95% (38/40) in the treated vs 92.3% (12/13) in the nontreated fertilized zygotes. We conclude that PAF treatment of rabbit spermatozoa prior to subzonal insertion improves fertilization rates and may be of clinical significance in assisted reproductive programs.

Selection of acrosome-reacted sperm with MH61-immunobeads.

Separation of acrosome-reacted viable sperm is an important problem in the analysis of sperm function and the micromanipulation of gametes such as the microinjection of sperm into ooplasm and subzonal sperm insertion. In the present study, we collected acrosome-reacted sperm selectively using paramagnetic immunobeads, referred to as MH61-beads, which attach themselves to the head of the acrosome-reacted sperm and are recovered by a magnet. The maximum number of sperm was recovered by the MH61-beads when 3 x 10(5) sperm/100 microliters and 6 x 10(5) MH61-beads were coincubated for 1 hour. The recovery of sperm was significantly correlated with the percentage of the acrosome reaction in the capacitation medium. After incubation for 12 hours, 2.4-9.0 x 10(4) sperm could be collected, and the percentage of viable and acrosome-reacted sperm was 28-60%. To improve the recovery of the acrosome-reacted sperm, we utilized follicular fluid, which induces the acrosome reaction. Addition of follicular fluid to the capacitation medium at a final concentration of 20% induced the acrosome reaction and increased the recovery of acrosome-reacted sperm significantly. In conclusion, separation of acrosome-reacted sperm by the MH61-bead-binding method could become a useful technique in research of sperm function and gamete micromanipulation.

Micromanipulation of human gametes for assisted fertilization

The techniques of partial zona dissection, subzonal sperm insertion, and intracytoplasmic sperm injection have been clinically employed to alleviate aberrations of human gamete interaction. This article reviews the current status of assisted fertilization technology and its relevance in treating male infertility. Indications for the application of each technique and factors affecting outcome are discussed.

Micromanipulation of human gametes for assisted fertilization

The techniques of partial zona dissection, subzonal sperm insertion, and intracytoplasmic sperm injection have been clinically employed to alleviate aberrations of human gamete interaction. This article reviews the current status of assisted fertilization technology and its relevance in treating male infertility. Indications for the application of each technique and factors affecting outcome are discussed.

Fertilization failure after subzonal sperm insertion associated with defective fusional capacity of acrosome-reacted spermatozoa

A case of an isolated impairment of the ability of acrosome-reacted spermatozoa to fuse with the oocyte is reported. This impairment was associated with fertilization failure after subzonal sperm insertion even though elevated numbers of spermatozoa were inserted per oocyte. This observation shows the need for further studies into the relationship between the acrosome reaction and the sperm-oocyte fusion to better understand the advantages and the limitations of subzonal sperm insertion in different types of sperm pathology.

Subzonal sperm insertion with aged human oocytes from failed in-vitro fertilization attempts: fertilization results and some applications.

This study was undertaken to assess the potential of aged human oocytes from failed in-vitro fertilization attempts as a model for the study of fertilization events after subzonal sperm insertion (SUZI). Criteria of aged oocyte suitability for this purpose were (i) the absence of nuclei, (ii) the presence of a polar body, (iii) the absence of cell division or fragmentation, (iv) marked ooplasmic contraction in hyperosmotic medium, and (v) rapid ooplasmic relaxation after returning into normo-osmotic medium following the micromanipulation. Micro-injection techniques were essentially the same as for SUZI with fresh oocytes. Oocytes that fused with the micro-injected spermatozoa developed pronuclei of typical internal structure. However, the number of pronuclei was often higher than that theoretically expected if each sperm nucleus incorporated into the oocyte gave rise to a single pronucleus. Thus, the use of aged oocytes implies the need for a specific method to assess the frequency of fusion in sperm samples examined. The results suggest that the method described here can be applied in a preliminary diagnostic test before a therapeutic SUZI attempt and in studies aimed at the optimization of sperm treatment protocols to increase the fusion capacity of subzonally inserted spermatozoa.

Medical abortion after in vitro assisted fertilization

Subzonal sperm insertion is an assisted insemination procedure to enhance IVF in patients with severe male factor. We report a lethal fetal abnormality that occurred in a SUZI-obtained pregnancy. It is essential that such cases be extensively collected to assess the feasibility of using this method in patients with severe semen defects.

A new rapid method of producing microneedles for subzonal sperm insertion

A new rapid method of producing microneedles for subzonal sperm insertion

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 semen parameters. We compared two sperm preparation methods (mini-Percoll and conventional swim-up) for efficiency of recovery after preparation and for fertilization rates after IVF, and compared the suitability of 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 significantly more motile spermatozoa from the same semen sample compared to the swim-up method. More oocytes were fertilized after spermatozoa were prepared by the mini-Percoll technique. An increased number of spermatozoa recovered from an ejaculate led to an improvement in the quality of spermatozoa in the insemination droplet. Subsequently, when using the PZD technique, the fertilization rate increased when there was a higher number of spermatozoa in the patient's ejaculate. When comparing the two micromanipulation techniques, SUZI provided patients with oligoasthenzoospermia (i.e. < 10 x 10(6) spermatozoa/ml and 40% motility) with a higher chance of obtaining 2-pronculeate eggs.

Analysis of factors contributing to success in a program of micromanipulation-assisted fertilization

To determine factors important to clinical success in micromanipulation-assisted in vitro fertilization (IVF). Procedures invoked in two separate series of micromanipulation-assisted IVF cycles, one unsuccessful (series I) and the other successful (series II), were compared in an effort to identify changes that led to clinical success. University-based IVF clinic. In both IVF series involving micromanipulation, patients consisted of infertile couples who fit any of five categories of male-factor related infertility. The female patients underwent controlled hyperstimulation for oocyte retrieval and the oocytes were inseminated normally or were subjected either to partial zona dissection or subzonal sperm insertion to assist fertilization. Results in all groups were compared between the two patient series. In the successful series II, a noticeable improvement in fertilization rate and embryo quality was observed compared with series I. A significant increase in the percentage of patients reaching embryo transfer, the pregnancy rate per transfer, and the pregnancy rate per retrieval were noted in series II; a 25% ongoing pregnancy rate per retrieval was observed overall in this successful group, with "ongoing" defined as manifestation of at least a fetal sac on ultrasound with no detectable problems. Patients with a mixed transfer of embryos derived from manipulated and normally inseminated oocytes had a 75% rate of pregnancy per transfer in series II. Differences between the two series could not be attributed to patient selection or biases in selection of oocytes relegated to micromanipulation. However, oocyte handling, micromanipulation, and culture protocols differed significantly between the two series in that temperature and pH of oocytes was better controlled, and micromanipulation time was minimized in series II. Success in micromanipulation depends on maintenance of the oocyte in a stable and supportive environment throughout the micromanipulation procedure. It is also important to minimize trauma to the eggs by performing micromanipulation rapidly and with minimal distortion of the egg. Patients with a poor fertilization rate in standard IVF may experience a substantial increase in the likelihood of pregnancy when micromanipulation-assisted fertilization is performed on some eggs.

Micromanipulation of gametes and embryos in preimplantation genetic diagnosis and assisted fertilization

Recent advances in micromanipulation and biopsy of gametes and embryos have made it possible to develop new approaches for early genetic diagnosis and prevention of genetic disease and for treatment of severe male-factor infertility. Preimplantation diagnosis of a number of X-linked and autosomal recessive disorders has been performed, using polar body sampling and blastomere biopsy, coupled with polymerase chain reaction. Blastocyst biopsy has also been performed in human embryos; however, there has been no clinical application so far. Existing data have not shown any detrimental effect of micromanipulation and biopsy involved in the preimplantation development of the human embryo. The existing experience on micromanipulation of gametes (zona-opening procedures, subzonal sperm insertion, and sperm microinjection into the ooplasm) has also demonstrated the clinical usefulness in assisted fertilization, suggesting a possible selective application of various micromanipulation techniques and their combinations in male infertility.

Microsurgical fertilization procedures: the absence of stringent criteria for patient selection.

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n = 200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n = 116 cycles) failed to fertilize in a previous cycle. Group B (n = 40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n = 94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.

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.

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.