CARTR Plus: the creation of an ART registry in Canada.

STUDY QUESTION: What is the status of fertility treatment and birth outcomes documented over the first 6 years of the Canadian Assisted Reproductive Technologies Register (CARTR) Plus registry? SUMMARY ANSWER: The CARTR Plus registry is a robust database containing comprehensive Canadian fertility treatment data to assist with providing evidence-based rationale for clinical practice change. WHAT IS KNOWN ALREADY: The rate of infertility is increasing globally and having data on fertility treatment cycles and outcomes at a population level is important for accurately documenting and effecting changes in clinical practice. STUDY DESIGN SIZE DURATION: This is a descriptive manuscript of 183 739 fertility treatment cycles from 36 Canadian clinics over 6 years from the CARTR Plus registry. PARTICIPANTS/MATERIALS SETTING METHODS: Canadian ART treatment cycles from 2013 through 2018 were included. This manuscript described trends in type of fertility treatment cycles, pregnancy rates, multiple pregnancy rates, primary transfer rates and birth outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Over the 6 years of the CARTR Plus registry, the number of treatment cycles performed ranged from less than 200 to greater than 1000 per clinic. Patient age and the underlying cause of infertility were two of the most variable characteristics across clinics. Similar clinical pregnancy rates were found among IVF and frozen embryo transfer (FET) cycles with own oocytes (38.9 and 39.7% per embryo transfer cycle, respectively). Fertility treatment cycles that used donor oocytes had a higher clinical pregnancy rate among IVF cycles compared with FET cycles (54.9 and 39.8% per embryo transfer cycle, respectively). The multiple pregnancy rate was 7.4% per ongoing clinical pregnancy in 2018, which reflected a decreasing trend across the study period. Between 2013 and 2017, there were 31 811 pregnancies that had live births from all ART treatment cycles, which corresponded to a live birth rate of 21.4% per cycle start and 89.1% of these pregnancies were singleton live births. The low multiple pregnancy rate and high singleton birth rate are associated with the increase in single embryo transfers. LIMITATIONS REASONS FOR CAUTION: There is potential for misclassification of data, which is present in all administrative health databases. WIDER IMPLICATIONS OF THE FINDINGS: The CARTR Plus registry is a robust resource for ART data in Canada. It provides easily accessible aggregated data for Canadian fertility clinics, and it contains data that are internationally comparable. STUDY FUNDING/COMPETING INTERESTS: There was no funding provided for this study. The authors have no competing interests to declare.

Transcriptional characteristics of different sized follicles in relation to embryo transferability: potential role of hepatocyte growth factor signalling.

STUDY HYPOTHESIS: We hypothesized that a better discrimination between follicles containing oocytes with high developmental competence and those containing oocytes with low competence, based on a combination of a follicle's size and transcriptomic signature, will provide a reliable method to predict embryonic outcome of IVF. STUDY FINDING: This study provides new insights on the impact of follicular size on oocyte quality as measured by embryonic development and demonstrates that medium follicles yield a better percentage of transferable embryos. WHAT IS KNOWN ALREADY: Although it is generally accepted that large ovarian follicles contain better eggs, other studies report that a better follicular size subdivision and a better characterization are needed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Individual follicles (n = 136), from a total of 33 women undergoing IVF, were aspirated and categorized on the basis of their follicular liquid volume (small, medium or large) and the embryonic outcome of the enclosed oocyte: poor or good development. Comprehensive gene expression analysis between cells from the different sized follicles was performed using microarrays and quantitative RT-PCR to find molecular markers associated with follicular maturity and oocyte developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis of embryonic outcome in relation to follicular size indicates that the medium-sized follicles category yielded more transferable embryos (35%) compared with the largest follicles (30%) (NS). Gene expression analysis revealed expression markers with significant (P < 0.05) discrimination between the poor development groups for all three follicle sizes, and good development medium-size follicles, including up-regulation of thrombomodulin, transforming growth factor, beta receptor II and chondrolecti, and those associated with hyaluronan synthesis, coagulation and hepatocyte growth factor signalling. LIMITATIONS, REASONS FOR CAUTION: These analyses were performed in a single cohort of patients coming from a single clinic and the biomarkers generated will require validation in different geographical and biological contexts to ensure their global applicability. WIDER IMPLICATIONS OF THE FINDINGS: Medium-size follicles seem to be the optimal size for a positive embryonic outcome and are associated with competence markers that may help in understanding the ideal differentiation status during late folliculogenesis. LARGE SCALE DATA: The data discussed in this publication have been deposited in The National Center for Biotechnology Information Gene Expression Omnibus database and are accessible through GEO Series accession number GSE52851. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) to M.A.S. There are no competing interests to declare.

Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding.

Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm-zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm-ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.

Intracellular pH regulation in human preimplantation embryos.

We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8-cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+)- and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range.

Inhibition by human embryos of mouse granulosa cell progesterone production: development of a sensitive bioassay.

Reproduction technologies could be improved by the development of methods to evaluate oocyte or embryo quality in a non-invasive, quantitative manner. Since human embryos secrete a factor that inhibits granulosa cell progesterone production, an interspecies bioassay was established to investigate whether the presence of this progesterone-inhibitory factor (PIF) in human embryo-conditioned (HEC) media is related to the health and developmental capacity of the embryos. Oocytes were microsurgically removed from oocyte-cumulus complexes isolated from superovulated mouse ovaries, and the oocytectomized complexes were cultured in HEC media in the presence of follicle stimulating hormone and testosterone. Progesterone accumulation in the media was determined by radioimmunoassay. Despite the potential limitations of very small volumes of HEC media to evaluate, and the need to freeze these media at the source, the bioassay was able to detect PIF activity in HEC media. Most embryos produced PIF activity, but the degree of inhibition was not correlated with the ability of oocytes to be fertilized, nor with embryo morphology or ability to cleave and develop after transfer. These results demonstrate that secretion of PIF by human embryos can be measured by this bioassay and that human PIF can inhibit murine granulosa cell steroidogenesis; however, PIF activity is not correlated with human embryo quality or developmental competence.

Lack of correlation between oocyte-corona-cumulus complex morphology and nuclear maturity of oocytes collected in stimulated cycles for intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the usefulness of morphology grading of the oocyte-corona-cumulus complex (OCCC) as a marker of oocyte nuclear maturity, fertilizability, embryo cleavage, and likelihood of pregnancy. DESIGN: Prospective cohort study. SETTING: Academic fertility center. PATIENT(S): Eighty-three infertile couples undergoing IVF-ET/intracytoplasmic sperm injection treatment. INTERVENTION(S): All patients underwent a long stimulation protocol of GnRH agonist therapy followed by hMG administration and transvaginal oocyte recovery. MAIN OUTCOME MEASURE(S): All OCCCs, oocytes, and embryos were assessed. The relation among OCCC morphology and the nuclear maturity of denuded oocytes, the fertilization rate, and embryo development to the cleavage stage were analyzed. RESULT(S): Of 909 OCCCs collected from 92 cycles, 2.5%, 4.2%, 79.9%, and 13.4% were prophase I, metaphase I, metaphase II, and degenerating, respectively. No statistically significant differences were found in the percentage of intact metaphase II oocytes, the fertilization rate, or the cleavage rate among complexes with different morphologic grades. The morphologic grade of the OCCCs of transferred embryos in the pregnant group was not different from that in the nonpregnant group. CONCLUSION(S): Most oocytes were in metaphase II at the time of retrieval after ovarian stimulation. However, no relation was observed between the OCCC morphologic grade and oocyte nuclear maturity, the fertilization rate, or embryo cleavage. These observations suggest that OCCC morphology grading is a poor marker of oocyte quality.

An allergic reaction following intrauterine insemination.

Intrauterine insemination is a common procedure used for the treatment of different causes of infertility. Adverse reactions associated with this procedure are very rare and usually the procedure is well tolerated by the patient. We report a case of an allergic reaction after intrauterine insemination. The patient developed fever, difficulty breathing and wheezing in both lung fields. Although a low concentration of penicillin in the medium was used, it caused a significant allergic reaction. When intrauterine insemination was performed in subsequent cycles with an antibiotic-free medium, no allergic reaction occurred, and the procedure was well tolerated by the patient. A careful allergy history is essential in patients pursuing infertility treatment where antibiotics are utilized. Patients who are known to be allergic to penicillin should have semen prepared by an antibiotic-free medium.

Sex ratio of babies is unchanged after transfer of fast- versus slow-cleaving embryos.

PURPOSE: A higher proportion of male offspring has been observed after transferring faster-developing embryos in a number of animal species. Therefore, we evaluated the correlation between the sex ratio of delivered babies and the cleavage stage of transferred embryos in a human IVF-ET program. METHODS: The sex of infants born (n = 104) after transfer of exclusively slower-cleaving < or = 3 cell (n = 41) versus exclusively faster-cleaving > or = 4 cell (n = 63) embryos was compared. Furthermore, all boys and girls resulting from IVF-ET (n = 213) were compared with respect to: the average number of cells in the embryos that were transferred, the embryo with the greatest number of cells in the cohort transferred and the percentage of embryos that were faster cleaving. RESULTS: Thirty seven percent (15/41) of infants resulting from the transfer of exclusively slower-growing embryos were girls and 38% (24/36) of the infants from the faster-growing embryos were girls (NS). The analysis all 213 babies born after 145 embryo transfer procedures did not suggest any differences in embryo cleavage rates in embryo transfers leading to male versus female infants. CONCLUSIONS: A greater number of boys born was not observed after transfer of faster-cleaving embryos as has been described in other animal species. The race to be male may not occur until later cleavage divisions or may not occur in the human embryo.

Role of a germ cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo fertilization.

Sulfolipid-immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077-1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm-egg binding assay with in vitro-capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm-zona pellucida interaction. In this study, using the in vitro sperm-egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the in vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti-SLIP1 IgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum IgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining.

Effects of human sera and human serum albumin on mouse embryo culture.

Human proteins normally used to supplement human in vitro fertilization-embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage. These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin. Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation. Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage. Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients. Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies. Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard. These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.

Preimplantation embryo development and serum steroid levels in immature rats induced to ovulate or superovulate with pregnant mares' serum gonadotropin injection or follicle-stimulating hormone infusions.

Follicular stimulation protocols using pregnant mares' serum gonadotropin (PMSG) or a follicle-stimulating hormone (FSH) preparation were compared to evaluate the yield and quality of embryos obtained from immature rats. Rats received a superovulatory dose of PMSG (40 IU), a nonsuperovulatory dose of the same gonadotrophin (4 IU), or a continuous s.c. infusion over a 72-h period with a purified FSH preparation containing an optimum ratio of luteinizing hormone (LH): FSH (FSH-hCG). The females were caged with fertile males on the evening of the 3rd day of gonadotropin treatment and scored for the occurrence of mating on the next morning; subgroups were killed on days 1-4 of pregnancy. High fertilization rates were observed in rats treated with 4 IU PMSG (84.1%) and in rats infused with FSH-hCG (91.0%); however, a much lower fertilization rate was observed following treatment with 40 IU PMSG (41.5%). From median ovulation rates of 9 and 79 in rats treated with 4 IU PMSG and in rats infused with FSH-hCG, medians of 8 and 69 embryos, respectively, were recovered from reproductive tracts flushed on day 4 of pregnancy, from which 75% were morulae or blastocysts; in contrast, from a median ovulation rate of 42.5, a median of only 12 embryos was recovered on day 3 of pregnancy following superovulation with 40 IU PMSG of which 80% were degenerate ova. Serum steroid profiles during the first 4 days of pregnancy differed significantly among treatment groups, the major differences being in substantially elevated levels of estradiol and androgens on days 1-3 in rats receiving the high (40 IU) dose of PMSG. Levels of these steroids in rats superovulated with the FSH-hCG infusion regimen were only marginally elevated above levels observed in rats treated with the low (4 IU) nonsuperovulatory dose of PMSG. Consistent with high ovulation rates, serum progesterone levels rose to considerably higher levels during the period in both superovulated groups than in animals receiving the low, nonsuperovulatory dose of PMSG. This work describes a novel method to superovulate rats (FSH-hCG) leading to high yields of normally developing embryos at all preimplantation stages and illustrates the close association between high yield of embryos and low levels of circulating androgens and estradiol-17 beta during the preimplantation period.

Uptake of an oviductal antigen by the hamster zona pellucida.

Using an antiserum raised against hamster oviductal zona pellucida, we observed specific immunogenic components of the reproductive tract on the zonae of oviductal eggs and in oviductal fluid. Results of immunohistochemical studies suggested that these oviductal components may originate from epithelial cells of the isthmus and, to a lesser extent, of the ampulla and fimbria. The oviductal immunogenic components have also been observed within the bursal cavity, which contains the ovary. These observations suggest that these oviductal components may play an important role in the first steps of the hamster reproductive process.

Formation of the hamster zona pellucida in relation to ovarian differentiation and follicular growth.

Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.

Preparation and properties of antibodies doubly labeled with tritium and fluorescein.

A method was developed to prepare doubly labeled antibodies whereby fluorescein-labeled antibodies were reacted with N-[ethyl-2-3H]ethylmaleimide. This tritiated conjugate exhibited the same immunoreactivity as the non-derivatized fluorescein-labeled antibody. This method provides a marker for immunocytochemical studies which takes advantage of the rapidity of fluorescent tracing combined with the stability and precision of the radioautography technique.

The cellular origin of the zona pellucida antigen in the human and hamster.

The origin of the zona pellucida in follicles at different stages of maturation was studied in the hamster and human by indirect immunofluorescence microscopy using a specific anti-hamster zona pellucida serum. The earliest detection of the zona pellucida material occurred in the ooplasm of oocytes in primordial follicles. In primary follicles, the fluorescence was localized at the periphery of the oocyte. In secondary and in mature follicles a deposition of fluorescent material was visualized on the inner and outer regions of the zona pellucida. Follicular cells, other than the oocyte, failed to exhibit a fluorescent reaction with the antizona pellucida serum. It is concluded that the oocyte is an important source of the antigenic material of the zona pellucida of hamster and human ova.