Thomas B. Pool

Successful in vitro fertilization and embryo transfer in cynomolgus monkeys.

We have started an in vitro fertilization program in cynomolgus monkeys in an effort to develop an appropriate animal model to improve our knowledge of early embryonic development. In 16 of 25 animals treated with menopausal gonadotropins, preovulatory follicles developed. Follicular aspiration was performed at laparotomy after human chorionic gonadotropin injection. A total of 299 follicles were aspirated, and 251 oocytes were recovered. Oocytes were cultured in 1 ml of growth medium or 100 microliter droplets of medium under mineral oil. Semen samples were obtained by electroejaculation, and the oocytes were inseminated 4 to 24 hours after aspiration. Culture under mineral oil significantly increased the fertilization and cleavage rates. Of 68 embryos produced, 24 have been used in 10 embryo transfers, resulting in two pregnancies.

High continuing pregnancy rates after in vitro fertilization-embryo transfer using medium supplemented with a plasma protein fraction * containing α- and β-globulins

Objective To evaluate both the laboratory and clinical outcomes after IVF-ET using culture media supplemented with a plasma protein fraction (PPF, Plasmatein; Alpha Therapeutics, Los Angeles, CA) containing albumin and significant amounts of α - and β -globulins. Design One-year clinical trial of a PPF with high globulin content as a medium supplement during IVF, embryo growth, and ET. Setting Fertility Center of San Antonio, a private, office-based center for assisted reproduction. Patients Ninety-eight couples, with women ranging in age from 26 to 46years, undergoing 103 ovum retrievals for IVF-ET as treatment for infertility because of tubal factor, endometriosis, anovulation, uterine or cervical factor, male factor, and unexplained causes. Main Outcome Measures Fertilization rate, zygote cleavage rate, clinical pregnancy rate (PR), continuing PRs, and implantation rates. Results Supplementation with PPF in insemination, growth and transfer medium resulted in a clinical PR of 41.5% per transfer with continuing PRs of 35.2% per retrieval, 37.2% per patient, and 38.7% per transfer. Conclusions A PPF containing significant amounts of α - and β -globulins can serve as an effective protein supplement to IVF medium, with outcomes manifested as high continuing PRs. These data indicate a potential role for glycoprotein components of serum in supporting healthy embryo growth in vitro, although the mechanism may relate more to the general physicochemical properties of this fraction than to the actions of a specific component.

The principal variables of cryopreservation: solutions, temperatures, and rate changes

OBJECTIVE To describe several fundamental variables that influence ultimate survival of oocytes and embryos when they are cryopreserved. DESIGN The literature describing fundamental and applied aspects of cryobiological variables that determine the responses of oocytes and embryos has been reviewed. CONCLUSION(S) When oocytes and embryos are to be cryopreserved, they are suspended in a solution of one of several low-molecular-weight solutes. The permeability of oocytes and embryos to these various low-molecular-weight compounds differs. These differences determine how these compounds are taken up by cells. That, in turn, influences how these compounds act to protect cells against damage when the cells are subjected to cryopreservation. Because of those protective effects, the compounds are referred to as cryoprotective additives. Another principal variable that influences oocyte and embryo survival is the rate at which the cells are cooled to subzero temperatures. After being stored for some time at -196°C in liquid nitrogen, the cryopreserved oocytes and embryos are warmed to liquefy the medium. The rate at which the specimens are warmed is at least as important, if not more important, in determining the ultimate survival of the oocytes and embryos. The effects of these physical variables on cell survival also are described.

Embryo cryopreservation in cynomolgus monkeys

An improved knowledge of cryopreservation of primate embryos will have important research and clinical application. Fifty-six 4- to 8-cell in vitro fertilized embryos were frozen in HEPES-buffered Tyrodes solution containing 1.5 M dimethylsulfoxide (DMSO) and cooled at the rate of 0.3 degrees C/minute to -39 degrees C before being transferred into liquid nitrogen. Embryos were rapidly thawed at room temperature for 2 minutes. DMSO was diluted with medium in three steps at 5-minute intervals. Of the 56 embryos, 39 (70%) were classified as viable on the basis of surviving the freezing process with greater than 50% of their blastomeres intact. Twelve of the 39 embryos were cultured overnight, and 11 cleaved at least once. Twenty-five embryos were transferred to nine synchronized, unstimulated recipient monkeys 24 to 48 hours after ovulation. Three pregnancies (33.3%) resulted from the nine transfers.

A simple approach to intracytoplasmic sperm injection

OBJECTIVE To describe a simple injection apparatus and method for performing intracytoplasmic sperm injection in a clinical IVF program. DESIGN A prospective clinical trial of intracytoplasmic sperm injection. SETTING A private office-based fertility program. PATIENTS Five couples undergoing IVF-ET with intracytoplasmic sperm injection as a treatment for male factor infertility. INTERVENTIONS Intracytoplasmic sperm injection was performed at room temperature (23.5 to 24.5 degrees C) in a simple zwitterion-buffered medium. MAIN OUTCOME MEASURES Fertilization rates, cleavage rates, clinical pregnancy rates, implantation rates. RESULTS Intracytoplasmic sperm injection was performed on 44 fresh oocytes from five patients. Twenty-three oocytes fertilized (52.3%) and 22 zygotes cleaved (95.7%). Three of five patients became pregnant (60%), resulting in the live birth of one normal male infant, one continuing singleton pregnancy, and one continuing twin gestation (46XX, 46XY). The implantation rate was 23.5%. CONCLUSION Intracytoplasmic sperm injection can be performed successfully in a simple medium at room temperature using commercially available microtools.

Sperm retrieval and concomitant tumor resection in azoospermic men with congenital adrenal hyperplasia and bilateral testicular adrenal rest tumors: a case report

PurposeThe objective of this study was to offer a new treatment approach for sperm retrieval simultaneously with tumor resection in azoospermic men with congenital adrenal hyperplasia (CAH), orchialgia, and bilateral testicular adrenal rest tumors (TARTs) who fail to respond to medical treatment.MethodsThis is a retrospective chart review from a couple’s fertility center.ResultsBetween May 2013 and May 2015, two azoospermic men with CAH and bilateral TARTs, with orchialgia, and desire to conceive underwent bilateral TART resection in the same surgical setting as sperm retrieval after remaining azoospermic with normalization of gonadotropins with treatment with human chorionic gonadotropin (hCG). Both men had adequate sperm retrieved for in vitro fertilization/intracytoplasmic sperm retrieval (IVF/ICSI) at the time of bilateral TART resections. They had complete TART resections with resolution of orchialgia. The wife of one patient had a successful pregnancy with use of retrieved sperm resulting in a live birth, and the sperm from the other man is cryopreserved for future use.ConclusionsIt is feasible to perform successful sperm retrieval simultaneously with TART resection in azoospermic men with CAH after medical treatments with persistent azoospermia, rather than subjecting these men to multiple invasive procedures.

New Concepts in Regulation of the Lifespan of Human Diploid Fibroblasts in Vitro

It was the initial observations of Swim and Parker (1957) on the longevity of human fibroblasts in cell culture, later confirmed and extended by Hayflick and Moorehead (1961) and Hayflick (1965), that have given rise to the discipline of cytogeronotology. That human embryonic fibroblasts demonstrate a finite lifespan in vitro has been taken as evidence for, and developed as a model system for, aging at the cellular level by a multitude of investigators following the pioneering work of Hayflick. Possibly no other single subdiscipline of cellular biology is more rampant with hypotheses offered to explain a given phenomenon than is cytogerontology. Although no single explanation has been uniformly derived or universally accepted regarding the mechanism of cellular senescence, the significance of the eventual answer has already been considered theoretically with respect to pathological implications (Goldstein et al., 1975; Timiras, 1975; Martin, 1979) and societal implications (Strehler, 1975). Some investigators question the extrapolation of data obtained from the classicalin vitro observations to the in vivo situation (see Orgel, 1973), while others question the phenomenon as being representative of cellular aging at all (see Bell et al., 1978). Whether the phenomenon (1) truly represents the normal sequence of events during cellular senescence, (2) is not aging but in reality is cellular differentiation, or (3) is simply an artifact of the conditions imposed upon cells in vitro will no doubt continue to be the subject of academic debate for years to come. That normal human diploid cells lose their ability to proliferate upon continuous subcultivation is not disputed.

Antimüllerian hormone as a predictor of good-quality supernumerary blastocyst cryopreservation among women with levels <1 ng/mL versus 1–4 ng/mL

OBJECTIVE To determine whether antimüllerian hormone (AMH) levels predict the availability of good-quality supernumerary blastocysts for cryopreservation. DESIGN Retrospective study. SETTING Two fertility centers. PATIENT(S) First fresh IVF cycles (n = 247) grouped as follows: 40 women <35 year old with AMH <1 ng/mL and 77 women with AMH 1-4 ng/mL; 62 women ≥35 year old with AMH <1 ng/mL, and 68 women with AMH 1-4 ng/mL. INTERVENTION(S) AMH level measured before IVF with ovarian stimulation protocols based on patient age and AMH level, including short gonadotropin-releasing hormone (GnRH) agonist, GnRH antagonist, or GnRH agonist microdose flare; supernumerary good-quality blastocysts cryopreserved on days 5 or 6 after retrieval. MAIN OUTCOME MEASURES(S) Supernumerary good-quality blastocysts for cryopreservation in relation to AMH levels. RESULT(S) Among women <35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocysts for cryopreservation between the groups with AMH <1 ng/mL and AMH 1-4 ng/mL (30.0% vs. 58.4%) when adjusted for age. Among women ≥35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocyst cryopreservation between groups with AMH <1 ng/mL and AMH 1-4 ng/mL (16.1% vs. 42.6%), when adjusted for age. CONCLUSION(S) Low AMH levels are associated with a statistically significantly lower likelihood of blastocysts for cryopreservation as compared with higher AMH levels. This effect was seen among women both <35 and ≥35 years of age. Patient counseling should include realistic expectations for the probability of good-quality supernumerary blastocysts available for cryopreservation.

Culture Media in IVF: Decisions for the Laboratory

Appreciation of the evolution of IVF culture media, highlighting salient discoveries that have led to the tremendous improvement over just a few short years ago, is instrumental in gaining insight into the complexities of gamete and embryo function. In turn, this knowledge brings understanding to the rationale behind current laboratory practices and aids in the ability to make informed decisions in regard to culture methods. Furthermore, discussion of impact of culture media on homeostatic regulation of gametes and embryos, focusing on key decisions made within the laboratory such as media type, macromolecule selection, and pH, further highlights their delicate nature, the need to minimize stressors, and ultimately provides insight into areas where future improvement can be made as we continue to strive for improvement in IVF success rates.

Optimization of Treatment Outcomes for Assisted Reproductive Technologies

The maximization of treatment outcomes is of the highest priority in the realm of Assisted Reproductive Technologies (ART). Couples and fertility centers have high expectations for successful in vitro fertilization (IVF) cycles. It is of paramount importance to preemptively identify and subsequently treat or correct any potential factors that may adversely affect the IVF cycle. In addition, in cases of recurrent implantation failure (RIF) or the downstream recurrent pregnancy loss (RPL), when couples have undergone a certain number of IVF cycles without establishing a potentially successful pregnancy, an understanding of the potential etiologies, evaluation, and whether or not possible treatment adjuncts are efficacious is important in terms of determining whether or not further troubleshooting is warranted. Clinical testing for the couple provides opportunities for troubleshooting prior to proceeding with the IVF cycle and includes a myriad of diagnostic tests which may lead to therapeutic management if any tests are out of normal range. Furthermore, various techniques within the embryology laboratory may optimize ART outcomes.