Dean Morbeck

Of the estimated 1.5 million men and women who were diagnosed as having cancer in 2010, approximately 10% are younger than 45 years. For these individuals, cancer treatment can be lifesaving but can permanently affect reproductive capacity. The American Society of Clinical Oncology has recommended that oncologists discuss the possibility of infertility with reproductive-age cancer patients and offer referral for fertility preservation consultation and therapy. Fertility preservation is an emerging field that offers treatment aimed at protecting future reproductive ability for individuals with cancer or other serious illnesses. Although fertility preservation strategies vary by patient age and sex, many allow patients to store gametes or reproductive tissues for potential future use to create offspring. As an emerging discipline, many questions remain about the role of fertility preservation. We performed a MEDLINE search from 1950 to June 2010 using the following MeSH terms: amenorrhea; antineoplastic agents; ovarian failure; premature; infertility, female; fertility preservation; infertility, male; adolescent and cancer; child and cancer; cryopreservation; and reproductive technologies, assisted. Studies considered for inclusion included those written in English and published before June 2010.

We aimed to determine whether embryo culture induces markers of cellular senescence and whether these effects were dependent on culture conditions. Murine blastocysts were derived in vitro and in vivo and assessed for 2 primary markers of senescence: senescence-associated β-galactosidase (SA-β-gal) and phosphorylated H2A.X (γ-H2A.X), the latter being a mark of DNA oxidative damage. Expression of senescence-associated genes p21, p16, and interleukin 6 (IL6) were also assessed. Compared with in vivo-derived blastocysts, in vitro embryos had high levels of SA-β-gal, nuclear γ-H2A.X, and p21 mRNA expression, indicating that a senescence-like phenotype is induced by in vitro culture. To determine the role of culture conditions, we studied the effect of oxygen (5 % vs 20 %) and protein supplementation on senescence markers. Blastocysts in reduced oxygen (5 %) had low levels of both SA-β-gal and γ-H2A.X compared with blastocysts cultured in ambient oxygen. Senescence markers also were reduced in the presence of protein, suggesting that antioxidant properties of protein reduce oxidative DNA damage in vitro. Elevated SA-β-gal, γ-H2A.X, and p21 suggest that in vitro stress can induce a senescence-like phenotype. Reduced oxygen during embryo culture minimizes these effects, providing further evidence for potential adverse effects of culturing embryos at ambient oxygen concentrations.

Mineral oil overlay microdrop is commonly used during in vitro fertilization (IVF) procedures. Though mineral oil appears homogeneous, it is an undefined product that can vary in quality. Here, we describe the history, chemistry, processing, and optimal use of mineral oil for IVF and embryo culture.

To compare sensitivity of inner cell mass (ICM) outgrowth assay and analysis of culture media amino acid turnover with the sensitivity of the human sperm motility assay (HSMA) and murine embryo assay (MEA) for detection of formaldehyde toxicity. Prospective in vitro study. University hospital-based infertility center. Murine embryos. The HSMA, MEA, and ICM outgrowth assays were performed with media containing 0-64-μM concentrations of formaldehyde. These assays were compared with dynamics of amino acid turnover in culture media. The lowest concentration of formaldehyde in culture media detected by each quality control assay. Sperm forward progression, but not motility, detected formaldehyde at a concentration of 32 μM. Sperm motility index identified formaldehyde toxicity at 64 μM, whereas blastocyst rates in the MEA were affected at 32 μM formaldehyde. Evaluation of ICM using outgrowth and grade detected 16 μM formaldehyde. Leucine turnover in culture media detected 64 μM formaldehyde in the amino acid assay. Inner cell mass outgrowth is a more sensitive bioassay than MEA and HSMA for the detection of formaldehyde in culture media. Amino acid metabolism may also provide a sensitive quality control measure for detection of formaldehyde.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)

The quality of in vitro culture conditions is a key component of a successful clinical embryology laboratory. Many, but not all, supplies used in the embryology laboratory are screened by the supplier with a bioassay. Embryology laboratories use a variety of approaches to verify the quality of mineral oil, protein, and disposables before clinical use; however, a best practice has not been determined. Some laboratories test every supply, even those already screened by the supplier, whereas other laboratories perform as little testing as possible. Despite screening by the supplier, recent reports of embryo toxicity, specifically with mineral oil, highlight that the integrity of the supply system has gaps. This review describes current bioassay quality control testing and discusses how it applies to screening of products with documented lot-to-lot variation.

Controlled-rate frozen and vitrified mature human oocytes were thawed or warmed and parthenogenically activated using ionomycin and 6-dimethylaminopurine to assess pronuclear development compared with fresh controls. This chemical activation model provides a useful tool for laboratories to assess proficiency before offering oocyte cryopreservation to their patients.