Partial Zona Dissection Research Articles

Unlocking the Potential of Assisted Hatching in Assisted Reproductive Technology: A Narrative Review.

The present study is set in the broad field of assisted reproductive technologies (ARTs) and examines various procedures under assisted hatching (AH). It also reviews their effects on implantation success rates. The primary emphasis is on explaining who has benefited and how many have benefited from these interventions. The most important factor determining the success rate of ART is implantation. To increase these rates, we use AH in our clinics to enhance each embryo's chances at life and substantially improve overall results. This comprehensive review includes various approaches, such as chemical-based measures (such as applying Tyrode's solution) and mechanical techniques (such as zona drilling and partial zona dissection). The individual techniques are carefully scrutinized, considering their mechanical detailing, methods of applying therapeutic effects, and the appropriateness of matching present social circumstances. The review begins by analyzing the basic nature of AH as a medium for embryo implantation and then focuses on how this detailed view reveals the advantages and drawbacks of various methods. Moreover, the articles discuss improvements in AH technology and many of the most modern technological developments that can help fine-tune ART issues. A major problem with these methodologies is that they involve serious risks and legal complications. However, a broad assessment of these topics allows us to understand their impact on fertility treatments. This review is written as a guidebook for physicians and researchers working in reproductive medicine. It compiles all current knowledge, providing literature to build on successes that will make breakthroughs possible in ART. Indeed, this is a valuable reference for guiding us in navigating complex AH procedures. It advances ARTs step by step toward perfection.

Equilibrium vitrification of oocytes using low concentrations of cryoprotectants

In order to make dry ice transportation of vitrified embryos practical, a near-equilibrium vitrification was developed using a cryoprotectant solution (EDFS10/10a), by which mouse embryos at various stages were vitrified in a near-equilibrium environment. EDFS10/10a consisted of 10% (v/v) ethylene glycol, 10% (v/v) Me2SO, 0.4 M sucrose and 24% (w/v) Ficoll PM70. This method exhibited the benefits of slow freezing and vitrification, with a low risk of osmotic injury. In this study, we investigated whether mouse oocytes are vitrifiable with EDFS10/10a in a highly dehydrated/concentrated state, and whether they can remain fertilizable and developing into embryos after vitrification. When mature mouse oocytes were vitrified in liquid nitrogen and after 4–28 days of storage at −80 °C, high survival rates were observed (88–99%). Vitrified and warmed oocytes were subjected to partial zona dissection and in vitro fertilized. The rate of 2-cell stage was 80–82%. Blastocyst formation rate was 55–70% which was similar to that of embryos derived from fresh oocytes. After the 2-cell embryos were transferred to recipient mice, the implantation and offspring rates did not differ significantly from those of embryos derived from fresh oocytes, indicating that vitrified oocytes retained the developmental ability. Therefore, it is possible to vitrify mouse oocytes in a near-equilibrium state using EDFS10/10a and conveniently transported using dry ice.

O-043 ICSI: the gamechanger in ART

Abstract On behalf of Neelke De Munck, Herman Tournaye and all colleagues BrusselsIVF (UZBrussel) and Vrije Universiteit Brussel (1980-2022) In the early years of ART, IVF was successful to alleviate female-factor or idiopathic infertility, yet limited results were obtained in male-factor infertility. In the 80s, assisted fertilization procedures using micromanipulation were introduced, i.e. zona drilling and partial zona dissection. As these procedures did not perform well, at BrusselsIVF we investigated subzonal insemination (SUZI) in mice and eventually we introduced SUZI in our clinic for couples with impaired sperm parameters. IRB approval was obtained in 1991 but required a prospective follow-up protocol of all pregnancies and children born. During SUZI, it occurred that sperm was injected in the oolemma, after which normal fertilization and embryo development was observed. The first pregnancy leading to a live birth in January 1992 was established when 11 oocytes had no fertilization after SUZI but one oocyte was fertilized after “failed SUZI” and was eventually transferred. In the following period, we were able to demonstrate that ICSI was more efficient than SUZI and from July 1993 we only applied ICSI in patients in need of assisted fertilization. We deliberately choose to share our experience widely with our peers and thereafter ICSI was performed in many centres worldwide and ICSI has been mushrooming. The 2017 ICMART World Report mentions an annual increase of ART cycles with 2 million cycles in 2017 resulting in 330.000 deliveries. The percentage of cIVF versus ICSI is very different depending on the region but on average 70% of cycles are ICSI. This indicates that ICSI is also widely used for non-male infertility. The occurrence of about 15% failed fertilization cycles after cIVF is often the reason for using ICSI. This is for sure the case in countries where only a limited number of cycles is reimbursed by social security or where no reimbursement is in place. ICSI proved to relieve infertility in couples with severely impaired characteristics of ejaculated sperm including cryptozoospermia. For these patients ICSI results were similar as cIVF for other indications. ICSI with epididymal or testicular sperm could also solve the problem in patients with obstructive azoospermia. Patients with non-obstructive azoospermia can also be helped if sperm cells can be found by testicular sperm extraction using conventional TESE or microTESE. However, the chance to have a child in NOA couples embarking for both TESE and eventually ICSI is much lower if expressed in terms of Intention to Treat: 13,4% in NOA patients and 10,1% in Klinefelter patients. Most of these couples will embark on treatment even after full counselling before considering the use of donor sperm. At BrusselsIVF, ICSI is also used on in-vitro matured oocytes following culture of immature cumulus-oocyte complexes collected from antral follicles in PCOS patients. For couples undergoing Preimplantation Genetic Testing with biopsy of day-3 cleaving embryos or day-5 or day-6 blastocysts, ICSI is still the preferred procedure in order to avoid contamination at the time of biopsy by remnants of sperm or cumulus cells when cIVF is used. From the start in 1980 we have invested in clinical, translational and fundamental research. As of now this research is carried out by more than 250 individuals from BrusselsIVF, the centre for reproductive medicine of the UZBrussel and different research units at the VUB. Research focuses on different aspects of infertility, medical genetics, human reproduction, early human development and human pluripotent cells.

Day 2 laser assisted hatching (AH) significantly improves implantation rates in fresh blastocyst transfers

The efficacy of assisted hatching has been widely debated. The variable results reported for AH are confounded by the numerous methods of performing AH, including mechanical partial zona dissection, acid tyrode’s, and more recently by laser assistance. Assisted hatching has most commonly been performed on Day 3 embryos. This study assesses if Day 2 laser assisted hatching can improve implantation rates for fresh blastocyst transfers. Prospective observational cohort. On the morning of Day 2 of culture, all embryos were sorted into groups (<4-cell, 4-cell and >4-cells). Those in the AH group underwent laser assisted hatching using an Octax laser (4.0ms) on the morning of Day 2 at the time of embryo check. All embryos were then cultured undisturbed until assessment on the morning of Day 5 when the highest quality embryo(s), based on morphology, were selected for embryo transfer. A total of 446 fresh Day 5 transfers between Jan 2016 - Mar 2019 were analyzed (244 transfers with AH and 202 transfers without AH). A total of 682 embryos were transferred in the 446 cycles (363 embryos in the AH group and 319 embryos in the non-AH group). Because 206 of the 446 transfers involved transfer of more than 1 embryo, a mixed model accounting for both fixed and random effects (i.e. repeated measurements) was used, with SAC modeled as a function of the fixed effects assisted hatching (AH), age, and body mass index (BMI). Day 2 assisted hatching is associated with successful implantation (p=0.036). As expected, age was negatively associated with implantation rate (p<0.0001). BMI was not. The R-square for the model was 0.52, and the variance component of the random effects (owing to multiple embryo transfers) was 0.082 (95% CI [0.046-0.11]).Tabled 1No AHWith AHTotal# Fresh Day 5 Transfers202244446Mean Maternal Age35.634.435.0Mean BMI26.827.026.9Mean # Embryos Transferred1.581.491.53# Embryos319363682# Sacs137194331Implantation Rate (%)42.9%53.4%48.5% Open table in a new tab Large data sets such as the SART database do not allow for evaluation of the impact of specific AH techniques. Certainly, any embryo handling and exposure has the potential to be detrimental to blastocyst development and implantation rates. Here, we show that Day 2 laser AH can lead to a significant increase in implantation rates from 42.9% without AH to 53.4% with AH. Day 2 embryos generally have larger perivitelline space which may allow for reduced peripheral laser exposure to the blastomeres. Further studies are needed to confirm if Day 2 laser AH confers benefit over Day 3 laser AH.

Increased Elasticity of the Zona Pellucida Affects Micromanipulation Procedures for Preimplantation Genetic Screening at the Cleavage Stage: Possible Role of Blastomere Alterations on Inconclusive or “No Results” Interpretations

Objective Occasionally, submission of blastomere(s) or trophectoderm cells for preimplantation genetic screening (PGS) may be interpreted as inconclusive (“no results”). The main reasons for those results include failure of DNA amplification, degraded DNA, or failure to load cell(s) into the biopsy tube. In a particular case at our clinic, blastomere biopsies were submitted for PGS and all embryos were labeled as “no results”. Interestingly, the zona pellucida (ZP) of all embryos was extremely elastic and blastomere displacement was difficult. We theorize that the high elasticity and extended micromanipulation procedures may have affected the DNA in such a way as to interfere with the accurate assessment/interpretation of results. Design Case Report. Materials and Methods A total of 43 oocytes were harvested at retrieval. Oocytes were inseminated via standard IVF or ICSI. Ten cleavage stage embryos with a minimum of 6 blastomeres were selected for biopsy/PGS screening. Assisted hatching (AH) via partial zona dissection (PZD; tridimensional) was performed prior to blastomere biopsy. Embryo biopsy was performed via blastomere displacement. Results The ZP of all biopsied embryos was highly elastic. In addition, the inner layer of the ZP appeared to be detached in certain areas for some embryos. Those embryos acquired an oval-elliptical shape. The lack of rigidity prevented generation of internal pressure inside the embryo, which is necessary to displace the blastomere(s). The ZP of each embryo had to be probed at different angles in order to displace the selected blastomere(s). The time required to harvest blastomeres was extended to approximately ≥5 min per embryo. Selected blastomeres were eventually isolated without evidence of damage to the remaining blastomeres. All embryos were labeled as “no results” after assessment by the genetic laboratory. The report and additional options were discussed with the patients, and it was decided to proceed with embryo transfer (D5) based on blastocyst morphology. Two high quality blastocysts were transferred, resulting in a singleton term pregnancy. The remaining viable embryos were considered for vitrification. Conclusions Zona pellucida elasticity and extended manipulation of embryos/blastomeres may result in a form DNA degradation that interferes with the accuracy or interpretation of results during genetic assessment. Further analysis of similar cases may shed light into the relationship between difficult biopsies and inconclusive PGS results. Disclosures Nothing to disclose.

28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO

For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.

Blastocyst collapsing and subsequent dissection of ¼ of zona pellucida before the embryotransfer significantly increase pregnancy rate in fresh cycles

Blastocyst’s inability to hatch out of zona pellucida in time may lead to asynchronization of embryo and endometrium development causing implantation failure. So the aim of the study was to evaluate the efficiency of blastocyst collapsing and partial zona dissection performed before the fresh embryotransfer to ultimately facilitate embryo hatching.

Sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts

Sucrose included in freezing medium causes embryos to shrink by losing intracellular water, so that intracellular ice formation is reduced and embryos can be frozen rapidly. Blastocyst consists of trophoblast and inner cell mass. It may be considered that the artificial shrunken blastocyst is not embryo but tightly packed somatic cells. This study was carried out to examine the necessary of sucrose addition to vitrification solution for vitrifying the artificial shrunken mouse blastocysts. Laboratory study using mice blastocyst. To investigate the toxicity of vitrification solutions and the availability of artificial shrinkage technique with ICSI pipette, blastocoel fluid was aspirated in the expanded mouse blastocysts using ICSI pipette and shrunken blastocysts were equilibrated in EBS1 and EBS2 for 1.5 min at room temperature, reapectively. Then the equilibrated blastocysts were exposed to vitrification solution for maximum of 20 sec. Two vitrification solutions with and without sucrose were prepared. Control and treatment were composed of 25%glycerol + 25% ethyleneglycol and 25%glycerol + 25%ethyleneglycero +0.5 mol/l sucrose, respectively. Assisted hatching was performed by partial zona dissection method using assisted hatching pipette after thawing procedure or rehydration. Rates of re-expanding and hatching were examined after culture for 6h. Re-expanding rate of mouse blastocyst exposed to VS(without and with 0.5 mol sucrose) were not different in G25E25(98%) and G25E25S0.5(92%) (P>0.05) and hatching rate was higher in G25E25(95%) than in G25E25S0.5(88%) but did not differ in two treatments (P>0.05). Re-expanding rate of mouse blastocyst vitrified in G25E25 and G25E25S0.5 was 95% and 90%, respectively (P=0.05), and hatching rate was higher in G25E25(90%) than in G25E25S0.5(76%) (P>0.05). Our results showed that sucrose needs not to be added in vitrification solution for freezing the artificial shrunken mouse blastocysts.

Assisted Reproductive Technology and Major Birth Defects in Western Australia

Assisted Reproductive Technology and Major Birth Defects in Western Australia

The effect of long zona dissection using ICSI pipettes for mechanical assisted hatching in vitrified-thawed blastocyst transfers

To investigate the effect of long zona dissection (LZD) compared with partial zona dissection (PZD) using ICSI pipettes for mechanical assisted hatching (AH) in vitrified-thawed blastocyst transfers. Prospective study. University IVF clinic. A total of 120 women ≦ 38years old undergone vitrified-thawed blastocyst transfers with LZD or PZD. The culture of all pronucleate embryos to the blastocyst stage and the selection of blastocysts ≧ grade 3BB (Gardner and Schoolcraft score), followed by vitrified-thawed blastocyst transfers with LZD (n = 60) or with PZD (n = 60) Complete hatching rates, implantation rates, pregnancy rates. At 5h after thawing, complete hatching rates of blastocysts were significantly higher in LZD group compared with PZD group, 52.4% vs. 31.8% (P = 0.001). Implantation and clinical pregnancy rates were significantly higher in LZD group compared with PZD group, 40.9% vs. 25.7% and 63.0% vs. 40.0%, respectively (P = 0.010, P = 0.011). LZD using ICSI pipettes for mechanical AH improves significantly complete hatching, implantation and pregnancy rates in vitrified-thawed blastocyst transfers.

Outcomes of preimplantation genetic diagnosis using either zona drilling with acidified Tyrode's solution or partial zona dissection

ObjectiveTo review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD).MethodsClinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared.ResultsApplication of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group.ConclusionThe mechanical zona dissection method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.

Celebrating ICSI's twentieth anniversary and the birth of more than 2.5 million children--the 'how, why, when and where'

The first major breakthrough for the alleviation of tubal infertility occurred in the last quarter of the twentieth century. Louise Brown was born on the 25 July, 1978 inOldham, England as a result of the pioneering work of Bob Edwards and the late Patrick Steptoe with the help of the late Jean Purdy. Bob Edwards received the ‘Nobel Prize for Physiology or Medicine’ for this work in 2010. In the early eighties IVF practice was gradually introduced in many countries and became a successful treatment for the alleviation of longstanding female-factor and idiopathic infertility. However, the hypothesis that bringing the male gametes closer to the oocytes would be an effective treatment in couples with reduced numbers of spermatozoa was not supported. As soon as sperm parameters were below a certain threshold, fertilization of oocytes was much reduced or failed completely. It became obvious that conventional IVF could not solve many cases of male-factor infertility. For couples with a very low sperm count or azoospermia it was necessary to resort to the use of artificial insemination with donor sperm (AID). As a consequence, several groups embarked on research aimed at assisting the fertilization process. Micromanipulation procedures were introduced. The two initial assisted fertilization procedures: zona drilling and partial zona dissectionwere not successful. Zona drilling involvedmaking a hole in the zona pellucida of an oocyte, whichwas then incubated in a sperm suspension. Zona drilling worked well in mice but not in humans. Partial zona dissection was introduced afterwards. A mechanical slit was made in the oocytes, which were then incubated in a sperm suspension. Fertilization was obtained by this method but when it occurred there was a similar percentage of monospermic and polyspermic fertilization. Although some pregnancies and births occurred, inconsistent results meant that partial zona dissection was not widely applied in the clinic. Around the same time a few case reports were published on the next assisted fertilization procedure: subzonal insemination (SUZI), a micromanipulation technique involving the insertion of a few spermatozoa between the zona pellucida and the membrane of the oocyte. We decided to embark on this approach for assisted fertilization. What did we do?

Vitrification of human single pronuclear oocytes following two approaches to polar body biopsy

The purpose of the present study was to investigate whether the size of the opening in the zona pellucida (ZP) of human single pronuclear (1PN) oocytes made by laser and partial zona dissection (PZD) techniques might interfere with the survival and subsequent development to blastocyst stage upon vitrification and warming. Moreover, the viability of these blastocysts was evaluated by comparing their total cell number (TCN) to the TCN of blastocysts developed from control non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using laser-assisted and PZD techniques, respectively. The size of ZP opening made by laser and PZD techniques did not interfere with survival (94.8% and 95.4%) or development to the blastocyst stage (27.8% and 26.1%). However, the TCN of laser-derived blastocysts was significantly lower than the TCN of blastocysts developed from non-vitrified control 1PN oocytes (48.7 ± 3.4 versus 70.8 ± 7.1, P < 0.028). The vitrification protocol used here is thus revealed to be an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the viability of blastocysts developed from laser-treated 1PN oocytes seems to be negatively affected by this method of biopsy. The purpose of the present study was to investigate whether the size of the opening made in the outer membrane (zona pellucida) of human single pronuclear (1PN) oocytes by mechanical dissection and laser techniques followed by polar body removal could hamper survival and development to the blastocyst stage upon vitrification and warming. Vitrification is a simple method of cryopreservation in which the intra- and extracellular solutions transform during cooling into a solid, glass-like substance. Moreover, in order to predict the viability of blastocysts developed from vitrified 1PN oocytes, their total cell number (TCN) was recorded and compared with the TCN of blastocysts developed from non-vitrified zona-intact 1PN oocytes. Prior to vitrification, a total of 97 and 88 1PN oocytes were subjected to polar body biopsy using the laser-assisted and mechanical method of dissection, respectively. Vitrification was also applied to 61 zona-intact 1PN oocytes. Following vitrification, no disparity in survival or further development to blastocyst stage was observed between either of the two biopsied groups and zona-inact 1PN oocytes, revealing that the vitrification protocol used here is an effective method for cryopreservation of 1PN oocytes following polar body biopsy. However, the TCN of blastocysts developed from laser-treated 1PN oocytes was considerably lower than the TCN of blastocysts developed from non-vitrified zona-intact 1PN oocytes ( n = 46) suggesting that the viability of blastocysts developed from laser-treated 1PN oocytes was negatively influenced by this method of biopsy.

In vitro fertilization/intracytoplasmic sperm injection for male infertility

Progress in the field of assisted reproduction, and particularly micromanipulation, now heralds a new era in the management of severe male factor infertility, not amenable to medical or surgical correction. By overcoming natural barriers to conception, in vitro fertilization and embryo transfer (IVF-ET), subzonal sperm insemination, partial zona dissection, and intracytoplasmatic injection of sperm (ICSI) now offer couples considered irreversibly infertile, the option of parenting a genetically related child. However, unlike IVF, which necessitates an optimal sperm number and function to successfully complete the sequence of events leading to fertilization, micromanipulation techniques, such as ICSI, involving the direct injection of a spermatozoon into the oocyte, obviate all these requirements and may be used to alleviate severe male factor infertility due to the lack of sperm in the ejaculate due to severely impaired spermatogenesis (non-obstructive azoospermia) or non-reconstructable reproductive tract obstruction (obstructive azoospermia). ICSI may be performed with fresh or cryopreserved ejaculate sperm where available, microsurgically extracted epididymal or testicular sperm with satisfactory fertilization, clinical pregnancy, and ongoing pregnancy rates. However, despite a lack of consensus regarding the genetic implications of ICSI or the application and efficacy of preimplantation genetic diagnosis prior to assisted reproductive technology (ART), the widespread use of ICSI, increasing evidence of the involvement of genetic factors in male infertility and the potential risk of transmission of genetic disorders to the offspring, generate major concerns with regard to the safety of the technique, necessitating a thorough genetic evaluation of the couple, classification of infertility and adequate counseling of the implications and associated risks prior to embarking on the procedure. The objective of this review is to highlight the indications, advantages, limitations, outcomes, implications and safety of using IVF/ICSI for male factor infertility to enable a more judicious use of these techniques and maximize their potential benefits while minimizing foreseen complications.

Micromanipulation Techniques in IVF

After the world witnessed groundbreaking early advances in assisted reproduction that utilized conventional in vitro fertilization in the human, more complex invasive techniques of oocyte and embryo manipulation were introduced to treat human subfertility. Amongst these were numerous micromanipulation techniques such as partial zona dissection (PZD; 1988), subzonal insemination (SUZI, 1988) and subsequently ICSI, which became the most powerful tool of assisted fertilization (1992). ICSI requires micromanipulation of both the male and the female gamete. Another important approach is laser-assisted opening of the zona pellucida at different stages of preimplantation development either; to assist hatching (1990) or to remove cells for preimplantation genetic diagnosis (1989), or fragments to restore the integrity of an embryo (1999). Sometimes even blastocysts considered for vitrification need further manipulation in order to artificially shrink the blastocoel which facilitates survival of expanded blastocyst (2002). Other methods for reproductive purposes, however, failed to gain acceptance in population, such as cytoplasm transfer (1997), nuclear transfer (1998) and cloning (1996), to name but a few.

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

Reduced Oocyte Damage Rates After ICSI With Blunted Needle

High survival and developmental rates of vitrified mouse zygotes following polar body biopsy

The effect of two different methods of polar body biopsy followed by vitrification on the survival and development to blastocyst stage of mouse zygotes was examined. Prior to vitrification, a total of 119 and 124 mouse zygotes were subjected to polar body biopsy using either laser-assisted or partial zona dissection (PZD) techniques, respectively. Vitrification was also applied to 122 zona-intact zygotes that served as a control group. Following vitrification, no differences in the rate of zygote survival (95.8%, 91.9% and 94.3%) or in the rate of development to expanded blastocyst stage (82.3%, 79.8% and 82.0%) were observed between the two groups of biopsied zygotes, or between the biopsied zygotes and control zygotes. The mean total number of cells comprising the blastocysts of controls (77.1 ± 4.7) was comparable to the mean cell number recorded in the laser (66.4 ± 4.7) and PZD (69.7 ± 5.3) groups. Blastocysts developed from laser-treated zygotes hatched much earlier than blastocysts developed from the control and PZD groups of zygotes ( P < 0.001). The data obtained in the present study demonstrate that, irrespective of the biopsy method used prior to vitrification, mouse zygotes survive and develop to blastocysts upon warming in proportions similar to those of non-biopsied zygotes.

IMPACT OF RECONSTRUCTION BY GERMINAL VESICLE TRANSFER ON NUCLEAR MATURATION OF DOMESTIC CAT OOCYTES

Cytoplasmic defects as a result of aging or cryopreservation can be circumvented in immature oocytes by transferring the germinal vesicle (GV) into a previously enucleated good quality oocyte. We are interested in oocyte reconstruction by GV transfer in felids to provide options for propagating rare domestic cat biomedical models as well as endangered species. Objectives of this study were to assess the impact of (1) cumulus cell removal, (2) treatments that allow viewing the GV, and (3) enucleation and fusion on subsequent oocyte meiotic resumption. In Experiment 1 (3 replicates), grade I immature cumulus-oocytes complexes (COC) collected from adult cat ovaries were denuded in 0.2% hyaluronidase and matured in vitro (28 h) alone (n = 56 total denuded oocytes; DO) or in co-culture (n = 52 DO with equal number of COC). Nuclear status of DO and intact COC was assessed by fixation in ethanol and Hoechst 33341 staining. The proportions of metaphase II stage (MII) oocytes was less (P < 0.05; ANOVA) when oocytes were denuded and matured alone (23.8 ± 2.7%; Mean ± SD) compared to the co-cultured DO (76.7 ± 4.3%) or intact COC (82.3 ± 3.1%). In Experiment 2 (3 replicates), oocytes were denuded and either treated to visualize the GV (n = 74; incubation in 7.5 μg/ml Cytochalasin B for 15 min at 38.5°C, centrifugation at 15,000 × g for 10 min, and exposure to 0.1 M sucrose for 10 min at 38.5°C) or not treated (n = 75; control). Nuclear status of treated and control DO was assessed after in vitro maturation (IVM) with intact COC (co-culture; ratio 1:1). Percentages of MII were similar (P > 0.05) between DO treated to visualize the GV (79.7 ± 3.5%) and controls (82.7 ± 4.6%). In Experiment 3 (4 replicates), oocytes were denuded and treated as described above. After partial zona dissection of DO and enucleation, GV surrounded by a small amount of cytoplasm (karyoplasts) were fused by two electrical pulses (1.8 kV/cm DC for 30 μs in 0.15 M sorbitol) with the same or different cytoplasts (n = 93 homoplasmic, 91 heteroplasmic reconstructions, respectively). Control oocytes (n = 85) were denuded, treated to visualize the GV and subjected to electric pulses alone. Nuclear status of reconstructed and control oocytes was assessed after IVM with intact COC (1:1). Percentages of fused oocytes after homo- (71.7 ± 6.3%) and heteroplasmic reconstruction (69.0 ± 4.2%) were similar (P > 0.05). The proportion of fused oocytes reaching MII after homo- (46.5 ± 2.2%) versus heteroplasmic (47.7 ± 1.4%) reconstruction also did not differ (P > 0.05), but was less (P < 0.05) than controls subjected to electric pulses alone (80.0 ± 3.5%). This is the first report in carnivores of successful oocyte reconstruction by GV transfer. Results clearly demonstrate that removing cumulus cells is detrimental to nuclear maturation, but can be circumvented by co-culturing DO with intact COC. Furthermore, treatments that allow viewing the GV have no impact on oocyte nuclear maturation. Although enucleation reduces the overall proportion of oocytes able to reach MII, neither electric pulses nor heteroplasmic reconstruction appear to have any influence on GV-cytoplasm interactions during meiotic resumption. (Supported by the National Institutes of Health K01 RR020045). (platform)

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

An improved mechanical technique for assisted hatching

Varied clinical outcomes of assisted hatching (AH) have been reported. We attempt to investigate whether the size of the zona opening created by AH is adequate for blastocyst hatching, and, if not, set up a new method to improve it. A new AH technique, long zona dissection (LZD), was established, and experiments were performed to compare the effects of different sizes of zona opening on complete hatching of blastocysts in mouse and human embryos in vitro. The LZD technique can create a long zona slit on early embryos, even blastocysts, with the slit size beyond two-thirds of zona diameter. Compared with three-dimensional partial zona dissection, LZD can significantly enhance the hatching speed and the rate of complete hatching of mouse blastocysts (93.9%). All (100%) human blastocysts completely hatched following LZD; however, when the slit size after AH was about two-fifths of zona diameter, more of the larger inner cell masses (ICM) became trapped by the zona opening during hatching than the smaller ICM (53.3 versus 12.5%, P = 0.01). Zona opening of moderate size following AH is inadequate for the completion of blastocyst hatching in vitro; in some cases, however, it can be significantly improved by LZD.