Joe B. Massey

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation

OBJECTIVE To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. DESIGN Case report. SETTING Private infertility clinic. PATIENT(S) A 28-year-old woman with tubal infertility undergoing IVF therapy. INTERVENTION(S) Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. MAIN OUTCOME MEASURE(S) Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. RESULT(S) None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partners sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. CONCLUSION(S) This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation.

Antiphospholipid antibodies and in vitro fertilization success: a meta-analysis

OBJECTIVE To evaluate whether the presence of antiphospholipid antibodies among women undergoing IVF affects the likelihood of IVF success. DESIGN A meta-analysis of seven eligible studies on antiphospholipid antibodies and IVF outcome. MAIN OUTCOME MEASURE(S) Odds ratios (ORs) and 95% confidence intervals (CIs) of an association between the presence of antiphospholipid antibodies and both clinical pregnancy and live birth from IVF. RESULT(S) There was no significant association between antiphospholipid abnormalities and either clinical pregnancy (OR 0.99; 95% CI 0.64-1.53) or live birth (OR 1.07; 95% CI 0.66-1.75) in IVF patients. CONCLUSION(S) The measurement of antiphospholipid antibodies is not warranted in patients undergoing IVF.

Partial dissection of the zona pellucida of frozen-thawed e human embryos may enhance blastocyst hatching, implantation, and pregnancy rates

The rate of successful implantation after replacement of frozen-thawed embryos in in vitro fertilization is commonly only 5% to 10% per embryo. A limiting factor may be inability of otherwise viable embryos to be released from the intact zonae pellucidae. Culture conditions and/or cryopreservation in in vitro fertilization may affect the zona and impair blastocyst hatching. Therefore opening of the zona by partial slicing by means of micromanipulation before replacement of early cleaved embryos may improve chances of eventual hatching (referred to as assisted hatching). In 65 thawed embryo replacement cycles methyl-prednisolone and antibiotics were given for 4 days mid cycle. Assisted hatching was performed in 32 cycles, with 33 cycles left as controls. Patients age, infertility, cycle supplementation, and number of thawed and replaced embryos did not differ significantly between the two groups. Rates of viable embryonic implantation were 16% (10/63) and 9% (6/64) in the assisted hatching and control groups, respectively. Group sizes need approximately to double before this trend toward improved implantation with the use of assisted hatching reaches statistical significance.

Cryopreservation of zygotes and early cleaved human embryos.

Zygotes and 2- to 5-cell human embryos were frozen in 1,2-propanediol and sucrose; results of the first 50 cycles (45 patients) are presented. A total of 41 zygotes (17 attempts at thawing) were thawed, resulting in six singleton clinical pregnancies (15% per embryo; 35% per cycle), of which three delivered, one aborted, and two are ongoing. Fifty-seven cleaved embryos were thawed in 33 other cycles, resulting in four singleton and one twin pregnancy (11% per embryo; 15% per cycle), of which four delivered and one is ongoing. Depending on the cell stage, 61% to 81% of embryos survived cryostorage, but 2-cell embryos did not implant. One fifth of cryoinjury was due to the formation of cracks in the zona pellucida. The incidence of implantation was not enhanced when more than one freeze/thawed embryo was replaced, most pregnancies being obtained from single embryo replacements. At least 8% more births are expected in addition to conventional in vitro fertilization methods when the current policy of replacing three fresh embryos and freezing the remainder using this technique is applied. This method will result in two to four times more pregnancies per spare embryo, compared with other cryopreservation methods using older embryos.

Development of antral follicles in cryopreserved cat ovarian tissue transplanted to immunodeficient mice

Ovarian cortex cryopreservation and xenotransplantation into immunodeficient mice represents a potential means for female germplasm conservation and an immediate model for investigation of folliculogenesis. The objectives of this study were to: (1) assess follicle survival after cryopreservation and transplantation of cat ovarian tissue into non-obese diabetic severely combined immunodeficient (NOD SCID) mice; and (2) evaluate the effects of gonadotropin treatments on follicular development in the transplanted tissue. Slices from the cat ovarian cortex were frozen and after thawing, transplanted under each kidney capsule of castrated male NOD SCID mice (eight xenografts in four mice). Sixty-two days after surgery, mice were randomly assigned (two per group) to gonadotropin-treated (eCG and hCG 88 h later) or control (saline-treated) groups. Twenty-four hours after the last injection, ovarian tissue was recovered and processed for histology. Fresh ovarian tissue from the same original source was similarly processed. Follicles were counted, measured, and classified as primordial, primary, secondary, or antral. Immunoreactive proliferating cell nuclear antigen (PCNA) stain was used to assess follicle viability. Microscopic examination revealed no evidence of necrosis or fibrosis. The grafts were well-vascularized, with follicles at all stages of development. Numbers of follicles in the transplanted tissue were markedly reduced compared to fresh tissue, with approximately 10% of follicles surviving freezing and transplantation procedures. Growing follicles positive for PCNA were found in all xenografts. Gonadotropin treatment did not alter the proportion of resting to growing follicles or mean follicle diameter by comparison with controls from untreated mice. By contrast, luteinization, but not ovulation, of antral follicles was observed only in grafts from treated mice. In summary, frozen-thawed cat ovarian cortex tissue not only survived xenotransplantation, it also contained follicles able to grow to antral stages. Exogenous gonadotropin treatment in this model resulted in luteinization of antral follicles but enhancement of follicular growth and ovulation did not occur.

Poor implantation of cryopreserved reinsemination-fertilized human embryos

OBJECTIVE To investigate whether a poor rate of implantation after in vitro fertilization (IVF) was due to poor embryonic/endometrial synchrony during the original IVF cycle, we have cryopreserved reinseminated-fertilized embryos for later more synchronous replacement after thawing. The chance of implantation of fresh reinseminated fertilized human oocytes is approximately one tenth that of timely fertilized embryos. STUDY DESIGN AND DATA: Retrospective study of 35 original oocyte collections in which initial normal fertilization was 47.3% (129/273 oocytes), with 49.6% fertilization (67/135) upon reinsemination. Of these, 70 initially fertilized and 67 reinsemination-fertilized embryos were cryopreserved, and 50 initially fertilized and all 67 reinsemination-fertilized embryos were subsequently thawed with 72% and 63% cryosurvival, respectively, (not significant). SETTING Private infertility clinic. RESULTS In 11 cycles, 23 thawed initially fertilized embryos (group A) were replaced with a 21.7% implantation rate per embryo; in 10 cycles, 13 initially fertilized and 12 reinsemination-fertilized embryos (group B) were replaced together with an 8% implantation rate; finally, in 16 cycles, 30 reinsemination-fertilized embryos (group C) were replaced with a 3.3% implantation rate (group A versus group C: P = 0.076). Comparison of clinical pregnancies between these groups was significantly different (6/11 versus 1/16; P = 0.0427). CONCLUSION Reinsemination-fertilized embryos survive freezing as well as initially fertilized embryos, but better embryonic/endometrial synchrony does not improve chances of their implantation.

The significance of platelet-activating factor and fertility in the male primate: a review

Abstract:  Since its discovery nearly 30 years ago platelet‐activating factor (PAF) has emerged as one of the more important lipid mediators known. PAF (1‐O‐alkyl‐2‐O‐acetyl‐sn‐glycero‐3‐phosphorylcholine) exists endogenously as a mixture of molecular species with structural variants of the alkyl moiety. PAF is a novel potent signaling phospholipid that has unique pleiotropic biological properties in addition to platelet activation. PAF also plays a significant role in reproduction and is present in the sperm of a number of primate species. PAF content in squirrel monkey sperm is significantly higher during the breeding season than the non‐breeding season. PAF content in rhesus sperm has a significant relationship with sperm motility. PAF content in human sperm has a positive correlation with seminal parameters and pregnancy outcomes. The enzymes (lyso‐PAF‐acetyltransferase and PAF‐acetylhydrolase) necessary for PAF activation and deactivation are present in primate sperm. PAF‐acetylhydrolase may act as a ‘decapacitation factor’. Removal of this enzyme during capacitation promotes PAF synthesis increasing primate motility and fertilization. PAF also plays a significant role in the fertilization process, enhancing the fertilization rates of oocytes. Enhanced embryo development has also been reported in oocytes fertilized with PAF‐treated sperm. Exogenous PAF will also significantly improve primate artificial insemination pregnancy outcomes. PAF antagonists inhibit sperm motility, acrosome reaction, and fertilization thus suggesting the presence of receptors for PAF. The PAF‐receptor is present on primate sperm, with altered transcript levels and distribution patterns on abnormal cells. Whereas, the exact mechanism of PAF in sperm function and reproduction is uncertain, its importance in normal primate fertility is substantial.

Simultaneous Detection of Chromosomes X, Y, 13, 18, and 21 by Fluorescence In Situ Hybridization in Blastomeres Obtained from Preimplantation Embryos

Purpose.Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21.Methods:Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21.Results:Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant.Conclusions:The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.

Exposure of preimplantation embryos to platelet-activating factor increases birth rate.

AbstractProblem: Platelet-activating factor (PAF) plays a significant role in fertility. Preimplantation stage embryos produce PAF (ePAF) which is required for development. PAFs mechanism of action is receptor-mediated and its presence has been reported in the developing mouse and human embryo. Exposure of preimplantation stage mouse embryos results in higher implantation rates. However, the effect of such treatment on live-birth rates and birth weights has not been reported. Therefore, the objective the study was to determine the effect of exposing preimplantation mouse embryos to PAF on subsequent birth rate and weight. Design: Two-cell stage preimplantation stage mouse embryos exposed to PAF (10−7 M) for 15 min prior to intraoviductal transfer. Methods: Preimplantation stage embryos were recovered from eCG/hCG primed BDF1 female mice. Embryos were exposed to synthetic PAF (10−7 M) for 15 min. PAF-treated embryos were transferred to the oviducts of pseudopregnant female CD-1 female mice. Superovulated and cultured BDF1 embryos not treated with PAF served as in vitro controls and naturally ovulated embryos with no collection/culture served as in vivo controls. Embryos were permitted to develop to term (18–21 days). The number of pups born per litter and litter weights subsequently were recorded. Results: A total of 160 BDF1 mouse embryos were collected, treated, and transferred (20 per CD-1 recipient) as described. There was a significant (P<0.05) increase in the number of pups born to the PAF treatment group (56/80; 70%) as compared to the control group (44/80; 55%). There was also a significant difference (P<0.05) in litter birth weights between the PAF (1.31 g/litter) and controls groups (1.25 g/litter). There was a significant difference (P<0.05) in birth weights between the PAF treatment group and the in vivo group (1.51 g/litter). There was a significant difference in birth weights between the in vitro control and in vivo groups (1.51 g/litter). There were no observational malformaties to pups born in any group. Conclusions: Brief exposure of preimplantation stage embryos to PAF will result in a significant increase of delivery rates (pups/litter) as well as birth weights. However, the increase of birth weight was significantly below that found naturally. Additional studies are warranted to elucidate the mechanism of PAFs action in the preimplantation stage embryo and subsequent uterine development.

Minimal stimulation with simplified monitoring for in vitro fertilization

PurposeThis retrospective, descriptive study was designed to determine the effectiveness of using clomiphene citrate in relatively high daily doses (100, 150, and 200 mg) with simplified monitoring for in vitro fertilization in a private office and surgical center. The self-selected study population comprised 109 women who were 25–42 years old, including 26 women whose husbands had mild male-factor infertility.ResultsDuring January 1992 through December 1993, 165 stimulation cycles resulted in 137 egg retrievals, 24 clinical pregnancies (17.5%), and 20 viable pregnancies (14.5%). Cycles that could not be completed (28/165 or 16.9% of all cycles) involved luteinizing hormone surges, insufficient follicles, or low estradiol levels. There were no pregnancies in patients who were 40 years or older or who received 200 mg daily doses of clomiphene citrate. The viable pregnancy rate among patients with male factor infertility was 7.7% (2/26). For non-male-factor infertility patients who were younger than 40, the viable pregnancy rate was 17.6% (18/102).ConclusionThe simplified monitoring method did not appear to compromise the results.