Elective Single Embryo Transfer in the Setting of Funded In Vitro Fertilization: Two Years' Experience in an Ontario Hospital-Based Fertility Clinic.

OBJECTIVE: The objective of this study was to assess data from a fertility clinic and identify differences in patient and cycle characteristics, clinical pregnancy rates, and multiple gestation rates before and after fertility treatment funding and a policy of elective single embryo transfer were instituted by the Ontario government to reduce multiple gestations arising from fertility treatment. METHODS: This study was a retrospective database review of clinic and embryology laboratory data for all patients undergoing in vitro fertilization (IVF) and intracytoplasmatic sperm injection (ICSI) cycles over a 4-year period. The investigators compared IVF and ICSI cycles before funding, from January 1, 2014 to December 31, 2015, with cycles after funding, from January 1, 2016 to December 31, 2017. RESULTS: The number of cycles performed over a 2-year period increased from 554 to 853, of which 76.2% were funded. Patient age, body mass index, and parity were similar before and after funding. Fewer patients receiving funded IVF or ICSI had had a previous cycle. Cycle cancellation rates were similar before and after funding; however, there were fewer embryo transfers per cycle start after funding (80.3% vs. 72.2%, P = 0.001). The clinical pregnancy rate was similar before and after funding (37.8% vs. 32.5%, P = 0.09), whereas the multiple gestation rate was significantly lower (13.1% vs. 3.5%, P = 0.001). CONCLUSION: Since the government of Ontario began funding IVF and ICSI cycles, more patients are accessing treatment, many for the first time. The clinical pregnancy rate was maintained, whereas multiple gestations were significantly reduced. These findings support the benefit of single embryo transfer in the context of funded IVF and ICSI and demonstrate the importance of government-funded assisted reproductive technology.

High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos.

Assisted reproductive technologies (ARTs) represent the best chance for infertile couples to conceive, although increased risks for morbidities exist, including imprinting disorders. This increased risk could arise from ARTs disrupting genomic imprints during gametogenesis or preimplantation. The few studies examining ART effects on genomic imprinting primarily assessed poor quality human embryos. Here, we examined day 3 and blastocyst stage, good to high quality, donated human embryos for imprinted SNRPN, KCNQ1OT1 and H19 methylation. Seventy-six percent day 3 embryos and 50% blastocysts exhibited perturbed imprinted methylation, demonstrating that extended culture did not pose greater risk for imprinting errors than short culture. Comparison of embryos with normal and abnormal methylation didn't reveal any confounding factors. Notably, two embryos from male factor infertility patients using donor sperm harboured aberrant methylation, suggesting errors in these embryos cannot be explained by infertility alone. Overall, these results indicate that ART human preimplantation embryos possess a high frequency of imprinted methylation errors.

Identification of WNT/beta-CATENIN signaling pathway components in human cumulus cells.

Signaling via the conserved WNT/beta-CATENIN pathway controls diverse developmental processes. To explore its potential role in the ovary, we investigated the expression of WNTs, frizzled (FZD) receptors and other pathway components in human cumulus cells obtained from oocytes collected for in vitro fertilization. Proteins were detected in cultured cells using immunofluorescence microscopy. Protein-protein interactions were analyzed by means of immunoprecipitation. WNT2, FZD2, FZD3 and FZD9 were identified but WNT1, WNT4 and FZD4 were not detected. WNT2 is co-expressed with FZD2, FZD3 and FZD9. Co-immunoprecipitation using WNT2 antibody demonstrated that WNT2 interacts with both FZD3 and FZD9, but only FZD9 antibody precipitated WNT2. We also identified DVL (disheveled), AXIN, GSK-3beta (glycogen synthase kinase-3beta) and beta-CATENIN. beta-CATENIN is concentrated in the plasma membranes. DVL co-localizes with FZD9 and AXIN in the membranes, but GSK-3beta has little co-localization with AXIN and beta-CATENIN. Interestingly, beta-CATENIN is highly co-localized with FZD9 and AXIN. CDH1 (E-cadherin) was also detected in the plasma membranes and cytoplasm, co-localized with beta-CATENIN, and CDH1 antibody precipitated beta-CATENIN. The results suggest that WNT2 could act through its receptor FZD9 to regulate the beta-CATENIN pathway in cumulus cells, recruiting beta-CATENIN into plasma membranes and promoting the formation of adherens junctions involving CDH1.

Factors affecting patients' attitudes toward single- and multiple-embryo transfer.

OBJECTIVE: To identify factors that influence patient decision making concerning embryo transfer. DESIGN: Prospective analysis. SETTING: In vitro fertilization unit at a tertiary-care, university-affiliated teaching hospital. PATIENT(S): Seventy-nine women and 53 men who were referred consecutively for IVF treatment. INTERVENTION(S): Provision of risk information about complications of twin pregnancy. MAIN OUTCOME MEASURE(S): Rated desirability of different transfer options and twin pregnancy, together with standardized measures of depression and infertility stress. RESULT(S): Women's initial preference for two-embryo transfer (2ET) was related to beliefs that the chance of pregnancy was higher with 2ET vs. elective single-embryo transfer and that the personal chance of twins was relatively likely with 2ET but was not related to a specific desire for twins. Providing risk information increased the desirability of elective single-embryo transfer and decreased the desirability of twin pregnancy among both men and women. CONCLUSION(S): Cautious patients, preferring transfer of fewer embryos, balance desires to maximize the chance of pregnancy with acceptance of risks associated with twins. Less-cautious patients may be motivated by beliefs about the influence of age, desires for, and likelihood of twin pregnancy. Information about risks may affect these groups differently and diverse patient motivations may require tailored information to ensure informed consent.

Development and investigation of a free and informed choice process for embryo donation to stem cell research in Canada.

OBJECTIVES: To develop and investigate a consent process that satisfies the Assisted Human Reproduction (AHR) Act and the Canadian Institutes of Health Research (CIHR) Stem Cell Guidelines, furthers free and informed choice, and fosters embryo donation to human embryonic stem cell (hESC) research. METHODS: Consultations were undertaken with an hESC scientist, in vitro fertilization (IVF) team members, and the ethicist-author of the CIHR Guidelines to review the AHR Act, the CIHR Stem Cell Guidelines, the established consent process for embryo donation at University Hospital, London Health Sciences Centre, the characteristics of patients appropriate for contact, and strategies for sensitive recruitment. Invitation-to-participate packages were sent to patients. RESULTS: Patients deemed appropriate for contact had indicated their intent to donate embryos to research, had embryos that had been cryopreserved for more than five years, had not received donor gametes, and had publicly listed addresses, with no suggestion of separation of the parties. Strategies developed to promote anonymity, confidentiality, and informed choice included a "firewall" between clinical and research teams and documents reiterating that, if embryos were donated, the woman would have to undergo additional IVF treatment to have a child. Of 40 couples contacted, only 22 agreed to donate embryos to the hESC study. One couple no longer wished to donate embryos to research, one package was returned as undeliverable, and no response was received from 16 couples. CONCLUSIONS: The consent requirements of the AHR Act and the CIHR Stem Cell Guidelines should be met. Consider delaying the request for final consent until a significant time after IVF treatment to ensure that patients no longer want their embryos for reproductive purposes and are free from perceptions of coercion. A consent process promoting free and informed choice, sensitive recruitment, and donation of embryos for hESC research should be developed by the Canadian professional bodies.

Leydig cell function in mice lacking connexin43.

Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.

Trafficking of circulating pro-NK cells to the decidualizing uterus: regulatory mechanisms in the mouse and human.

Natural Killer (NK) lymphocytes, strongly expressing CD56, become abundant in the human uterus three to five days after the mid-menstrual cycle surge in pituitary-derived luteinizing hormone (LH). The primary functions of LH are to initiate final oocyte maturation/ovulation and to contribute to decidualization of the uterine stroma. Decidualization is the transformation of estrogen-primed uterine stromal fibroblasts into large hormone-producing cells under the influence of progesterone (P4). Decidual CD56bright (dNK) cells are a distinct, transient, tissue-specific NK cell subset that undergoes proliferation, terminal differentiation, and then death prior to menses. If pregnancy occurs, dNK cells increase during first trimester, then decline and are virtually absent in late pregnancy. In mouse models, pregnancy-associated uterine NK (uNK) cells appear coincident with onset of decidualization during embryonic implantation. Murine uNK cells traffic from the circulation to the antimesometrial side of the uterus and migrate to the mesometrial side of each implantation site. Here they proliferate and are implicated in regulation of midgestation structural changes to major arteries supplying the placenta, before dying in late gestation. Emerging data indicate that interactions between lymphocytes and endothelial cells within the uterine microenvironment are mediated by classical molecules associated with lymphocyte trafficking in immune surveillance and in response to inflammation. Here, we review factors influencing NK cell trafficking to decidualizing murine and human uteri and the differentiation and functions of these cells within the uterus.

Human sperm sex hormone-binding globulin isoform: characterization and measurement by time-resolved fluorescence immunoassay.

CONTEXT: SHBG gene expression in human testis results in an SHBG isoform that accumulates in the sperm head. OBJECTIVE: The objective of this study was to further characterize the SHBG isoform in human sperm and to assess its biological relevance. DESIGN, SETTING, AND PATIENTS: A time-resolved immunofluorometric assay was established to measure SHBG isoform concentrations in sperm samples from patients and sperm donors attending in vitro fertilization clinics. RESULTS AND CONCLUSIONS: Molecular characterization of SHBG transcripts in human testis and sperm and biochemical analyses of the sperm SHBG isoform indicate that its smaller size compared with plasma SHBG is due to a lack of amino-terminal residues. The SHBG isoform is lost from sperm by one freeze and thaw cycle and during capacitation, which suggests it is located in or between the outer acrosomal and sperm plasma membranes. Sperm SHBG levels were proportional to the number of sperm analyzed and within assay variability in samples taken on different occasions from seven of nine individuals. Intra- and interassay variability (coefficient of variation) was 5.8 and 8.5%, respectively. Sperm SHBG levels ranged from 6-49 pm/10(6) sperm in 13 donor samples and did not correlate with serum SHBG levels. Sperm SHBG levels were lowest in fertile men and highest in patients with untreated varicocele, but these differences were not significant. Patients studied for couple infertility and those with surgically treated varicocele showed intermediate values. Sperm SHBG isoform levels correlate significantly with age and sperm motility and may influence sperm function in relation to male fertility.

Periovulatory increases in tissue homing potential of circulating CD56(bright) cells are associated with fertile menstrual cycles.

CD56(bright) lymphocytes appear in the uterus 3-5 d after ovulation coincident with the onset of stromal cell decidualization. Although the source of these uterine immune cells is not defined, a subset of blood CD56(bright) cells exhibits enhanced capacity to adhere to decidual vascular endothelium during the periovulatory period of menstrual cycles. In this study, the effects of early pregnancy on the adhesive capacity of CD56(bright) cells to bind uterine substrates were examined in a time-course study of 18 infertile women undergoing natural cycles before transfer of frozen/thawed embryos and 18 infertile women undergoing controlled ovarian stimulation. There were three pregnancies in the natural cycle group and seven in the hormone-stimulated cohort. Hormone levels, and number and quality of transferred embryos were similar between pregnant and nonpregnant cycles. However, the adhesive function of CD56(bright) cells increased before ovulation in hormone-treated women who became pregnant and before embryo transfer in naturally cycling women who became pregnant. This pattern of incremental adhesion, which was less frequently observed in unsuccessful cycles, suggests a role for NK cells in implantation. These results support the idea that temporal control of NK cell homing to the uterine microenvironment is a prerequisite to pregnancy.

Embryo donation: attitudes toward donation procedures and factors predicting willingness to donate.

BACKGROUND: The aim of the study was to assess infertile couples' attitudes toward the procedures of embryo donation (ED) and to identify factors predicting interest in donation. METHODS: Fifty-one couples who had received IVF treatment and had subsequently had embryos cryopreserved for >3 years were located and sent written information about the procedures for ED and possible implications of donation. A total of 49 couples agreed to participate in the study with 36 women and 31 men subsequently returning questionnaires describing their reasons for not claiming unused embryos and attitudes towards ED. RESULTS: Patients were supportive of donor screening procedures, but less comfortable sharing non-identifying information. Comfort levels declined as information became increasingly personal. Support for unconditional (i.e. the donation of embryos without conditions attached) and conditional (i.e. where couples could limit the donation of their embryos to persons/couples according to their preferences) models of donation was highly polarized and a substantial minority expressed strong opposition to each model. Willingness to donate was associated with greater comfort about disclosing personal information, a desire to know the outcome of donation and willingness to have future contact with a child, but not with current family size. CONCLUSIONS: Comfort in sharing information with a recipient couple is more important than acceptance of screening procedures, or attainment of family size goals in predicting willingness to donate embryos. Offering the option of conditional donation could increase the acceptability of ED for some patients.

A human sex hormone-binding globulin isoform accumulates in the acrosome during spermatogenesis.

Human sex hormone-binding globulin (SHBG) binds estradiol and testosterone with high affinity. Plasma SHBG is produced by hepatocytes, but the human SHBG gene is also expressed in the testis. Little is known about SHBG gene expression in the human testis, but human SHBG transcripts accumulate in a spermatogenic stage-dependent manner in the testes of mice containing an 11-kb human SHBG transgene. We have now found that human SHBG transcripts containing an alternative exon 1 sequence are located specifically in the testicular germ cells of these transgenic mice, whereas murine SHBG transcripts are confined to Sertoli cells. In addition, we have detected immunoreactive human SHBG in the acrosome during all stages of spermiogenesis in mice containing an 11-kb human SHBG transgene. Western blots of germ cell extracts from these transgenic mice and from human sperm indicate that the immunoreactive human SHBG in the acrosome composes electrophoretic variants, which are 3-5 kDa smaller than the major electrophoretic isoforms of human SHBG in the blood. This apparent size difference is due in part to differences in glycosylation of plasma and acrosomal SHBG isoforms. The function of the human SHBG isoform in the acrosome is unknown, but it binds steroid ligands with high affinity. This is the first demonstration that human SHBG transcripts encode an SHBG isoform that remains within a cellular compartment.