Vitaly A. Kushnir

Preimplantation genetic screening (PGS) still in search of a clinical application: a systematic review

Only a few years ago the American Society of Assisted Reproductive Medicine (ASRM), the European Society for Human Reproduction and Embryology (ESHRE) and the British Fertility Society declared preimplantation genetic screening (PGS#1) ineffective in improving in vitro fertilization (IVF) pregnancy rates and in reducing miscarriage rates. A presumably upgraded form of the procedure (PGS#2) has recently been reintroduced, and is here assessed in a systematic review. PGS#2 in comparison to PGS#1 is characterized by: (i) trophectoderm biopsy on day 5/6 embryos in place of day-3 embryo biopsy; and (ii) fluorescence in-situ hybridization (FISH) of limited chromosome numbers is replaced by techniques, allowing aneuploidy assessments of all 24 chromosome pairs. Reviewing the literature, we were unable to identify properly conducted prospective clinical trials in which IVF outcomes were assessed based on “intent to treat”. Whether PGS#2 improves IVF outcomes can, therefore, not be determined. Reassessments of data, alleged to support the efficacy of PGS#2, indeed, suggest the opposite. Like with PGS#1, the introduction of PGS#2 into unrestricted IVF practice again appears premature, and threatens to repeat the PGS#1 experience, when thousands of women experienced reductions in IVF pregnancy chances, while expecting improvements. PGS#2 is an unproven and still experimental procedure, which, until evidence suggests otherwise, should only be offered under study conditions, and with appropriate informed consents.

Hypoandrogenism in association with diminished functional ovarian reserve

STUDY QUESTION Is diminished functional ovarian reserve (DFOR) associated with low androgen levels? SUMMARY ANSWER Low androgen levels are associated with DFOR at all ages. WHAT IS KNOWN ALREADY Androgen supplementation via dehydroepiandrosterone (DHEA) has been reported to improve functional ovarian reserve (FOR); pregnancy rates in IVF cycles are associated with how well DHEA converts to testosterone (T); and androgen effects through the androgen receptor have been demonstrated in mice to beneficially affect early stages of follicle maturation. STUDY DESIGN, SIZE, DURATION In a controlled cohort study we investigated consecutive women presenting to our center with two forms of DFOR, premature ovarian aging/occult primary ovarian insufficiency (POA/OPOI) and physiologic diminished ovarian reserve (DOR). As controls for POA/OPOI patients, infertile women with normal age-specific FOR were recruited. PARTICIPANTS/MATERIALS, SETTINGS, METHODS The study involved 140 women with POA/OPOI, defined as age <38 years and abnormally low FOR by age-specific FSH and/or anti-Müllerian hormone (AMH), 166 women with DOR, defined as women age >40 years. Forty-nine control patients <38 years demonstrated normal FOR by FSH and/or AMH. In all three patient groups dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS), total testosterone (TT) and free testosterone (FT) at the time of initial presentation to our fertility center were assessed. In a small subgroup of women early morning cortisol levels were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE DHEAS marginally varied between the three groups (P = 0.04), with older women with DOR actually demonstrating higher levels than controls (P = 0.03). TT differed between the three groups more profoundly (P = 0.005), with women with POA/OPOI demonstrating significantly lower levels than controls (P = 0.009). Adjustment for body mass index, age and race in principle maintained observed differences in TT between groups, while adjustment for FMR1 (fragile X mental retardation 1) genotypes/sub-genotypes eliminated all differences. All three patient groups demonstrated low morning cortisol levels. LIMITATIONS, REASONS FOR CAUTION While results support lower androgen levels in women with DOR, and even more so in women with POA/OPOI, presented data should be viewed as preliminary, considering the known variability of androgen levels and the small number of women in whom morning cortisol levels were available. WIDER IMPLICATIONS OF THE FINDINGS Especially at young ages DFOR appears associated with significant hypoandrogenism (low T) in comparison with young control patients with normal FOR, raising the question whether this hypoandrogenism originates in adrenals or ovaries. POA/OPOI, thus, phenotypically mimics the polycystic ovary syndrome, where similar questions arise, though in regard to hyperandrogenism. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the Foundation for Reproductive Medicine, a not-for-profit medical research foundation and intramural funds from the Center for Human Reproduction. N.G. and D.H.B are members of the Board of the Foundation for Reproductive Medicine. N.G., A.W. and D.H.B. received research support, lecture fees and travel support from a variety of pharmaceutical and medical device companies, none in any way related to the issues discussed in this manuscript. N.G. and D.H.B. are listed as co-inventors on two, already granted US user patents, which claim therapeutic benefits from DHEA supplementation in women with DFOR and DOR: both authors are also listed on additional pending patents in regard to DHEA supplementation and on pending patents, claiming diagnostic and therapeutic benefits from the determination of CGG repeats on the FMR1 gene. N.G. is the owner of the Center for Human Reproduction, where this research was performed.

Endocrine autoimmune diseases and female infertility

An increasing body of evidence suggests that immune-mediated processes affect female reproductive success at multiple levels. Crosstalk between endocrine and immune systems regulates a large number of biological processes that affect target tissues, and this crosstalk involves gene expression, cytokine and/or lymphokine release and hormone action. In addition, endocrine–immune interactions have a major role in the implantation process of the fetal (paternally derived) semi-allograft, which requires a reprogramming process of the maternal immune system from rejection to temporary tolerance for the length of gestation. Usually, the female immune system is supportive of all of these processes and, therefore, facilitates reproductive success. Abnormalities of the female immune system, including autoimmunity, potentially interfere at multiple levels. The relevance of the immune system to female infertility is increasingly recognized by investigators, but clinically is often not adequately considered and is, therefore, underestimated. This Review summarizes the effect of individual autoimmune endocrine diseases on female fertility, and points towards selected developments expected in the near future.

The status of public reporting of clinical outcomes in assisted reproductive technology

OBJECTIVE To assess the transparency of assisted reproductive technology (ART) surveillance reports published by the Centers for Disease Control and Prevention (CDC) and by the Society for Assisted Reproductive Technologies (SART). DESIGN Retrospective analysis. SETTING Private clinical ART and research center. PATIENT(S) We analyzed ART data for the years 2005-2010, which were reported under federal mandate to the CDC (818,927 completed cycles) and voluntarily to SART (812,400 initiated cycles). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Initiated cycles excluded from final outcome reporting were used to evaluate transparency. RESULT(S) Only SART, but not CDC, reported initiated cycles, allowing analysis of excluded cycles. Excluded cycles increased significantly from 3.3% to 7.4% between 2005 and 2010. By 2010, 13/341 (3.8%) ART centers accounted for 50% of excluded cycles, representing an average of 37.3% of their cycles. These 13 clinics reported significantly better pregnancy and cancellations rates than national averages and collectively increased by 19.9% their share of U.S. ART cycles. CONCLUSION(S) Our data indicate decreasing transparency in public ART reporting in the United States, likely due to changes in practice and reporting patterns. A few clinics accounted for the majority of excluded cycles, leading to improved reported clinical outcomes and increasing market share. CDC and SART should ensure that all ART clinics publicly report the outcomes of all initiated cycles including embryo-banking cycles. ART surveillance and quality of care may be improved by prospectively tracking the total reproductive potential of each initiated cycle.

A randomized clinical trial of endometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates

OBJECTIVE To investigate whether granulocyte colony-stimulating factor (G-CSG) affects endometrial thickness, implantation rates, and clinical pregnancy rates in routine, unselected IVF cycles. DESIGN Registered, individually randomized, two-group, parallel double-blinded placebo-controlled clinical trial. SETTING Academically affiliated private clinical and research center. PATIENT(S) 141 consecutive, unselected, consenting women with no history of renal disease, sickle cell disease, or malignancy who were undergoing IVF. INTERVENTION(S) Sealed, numbered, opaque envelopes assigned 73 patients to receive G-CSF (Filgrastim, Amgen, 300 μg/1.0 mL) and 68 to receive placebo (saline). MAIN OUTCOME MEASURE(S) Endometrial thickness, clinical pregnancy, and embryo implantation rates. RESULT(S) The mean age for the whole study group was 39.59 ± 5.56 years (G-CSF: 39.79 ± 5.13 years; placebo: 39.38 ± 6.03 years). Endometrial thickness statistically significantly increased over the 5-day observation period for the whole group by approximately 1.36 mm. The increase in the G-CSF group was not statistically significantly different from the control group. Statistical models looking at treatment effects on clinical pregnancy and implantation rates demonstrated no effect of G-CSF treatment. There were no adverse events for either treatment group. CONCLUSION(S) In normal IVF patients, G-CSF does not affect endometrial thickness, implantation rates, or clinical pregnancy rates. Because these results were obtained in an older patient population, they may not necessarily apply to younger women. CLINICAL TRIAL REGISTRATION NUMBER NCT01202656.

Is it time for a paradigm shift in understanding embryo selection

BackgroundEmbryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF.DiscussionWe here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients.SummarySince newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

Number of Embryos Transferred After In Vitro Fertilization and Good Perinatal Outcome

OBJECTIVE: To assess the association between number of embryos transferred and a measure of assisted reproductive technology success that emphasizes good perinatal outcome. METHODS: We analyzed assisted reproductive technology cycles initiated in 2011 that progressed to fresh embryo transfer among women using autologous oocytes and reported to the U.S. National Assisted Reproductive Technology Surveillance System (n=82,508). Percentages of good perinatal outcome (live birth of a term [at or after 37 weeks of gestation], normal birth weight [2,500 g or greater] singleton) were stratified by prognosis (favorable, average, less favorable), age, embryo stage (day 3, day 5), and number of embryos transferred. Differences in the percentages by number of embryos transferred were evaluated using Fisher’s exact test with Bonferroni correction. RESULTS: Among patients younger than 35 years with a favorable prognosis, chances of a good perinatal outcome were higher with transferring a single (compared with double) day 5 (43% compared with 27%) or day 3 embryo (36% compared with 30%). Likewise, a higher chance of a good perinatal outcome was observed with transferring a single day 5 embryo in patients 35–37 years old with a favorable prognosis (39% compared with 28%) or patients younger than 35 years old with an average prognosis (35% compared with 26%). A higher chance of good perinatal outcome was associated with transferring two (compared with one) day 3 embryos among patients aged 40 years or younger with an average prognosis or patients younger than 35 years old with a less favorable prognosis. CONCLUSION: The association between number of embryos transferred and the birth of a term, normal birth weight singleton is described. Among patients younger than 35 years of age undergoing in vitro fertilization with a favorable prognosis, the highest chance of good perinatal outcome is associated with a single embryo transfer. LEVEL OF EVIDENCE: II

Outcomes of Fresh and Cryopreserved Oocyte Donation

Outcomes of Fresh and Cryopreserved Oocyte Donation Use of oocytes donated for in vitro fertilization (IVF) has increased in recent years.1 Donated fresh oocytes traditionally have been used immediately, creating embryos for transfer into the uterus, with extra embryos being cryopreserved for later use. In January 2013, the American Society for Reproductive Medicine declared the technique of oocyte cryopreservation (egg freezing) no longer experimental, although it called for “more widespread clinic-specific data on the safety and efficacy of oocyte cryopreservation ... before universal donor oocyte banking can be recommended.”2 Based on data that IVF outcomes with cryopreserved and fresh donor oocytes are comparable,3 some IVF centers established frozen donor egg banks. However, data reflecting IVF outcomes in routine clinical practice with cryopreserved donor oocytes have not been published.

Systematic review of worldwide trends in assisted reproductive technology 2004–2013

BackgroundAssisted Reproductive Technology (ART) has undergone considerable changes over the last decade, with consequences on ART outcomes in different regions of the world being unknown.MethodsWe conducted a systematic review of published national and regional ART registry data to assess how changes in clinical practice between 2004 and 2013 have impacted outcomes in Australia and New Zealand, Canada, Continental Europe, the United Kingdom (U.K.), Japan, Latin America, and the United States (U.S.). The data reflect 7,079,145 total ART cycles utilizing both fresh and previously cryopreserved embryos from autologous oocytes that resulted in 1,454,724 live births. This review focused on the following measures: ART cycle volume, use of cryopreserved embryos, single embryo transfer (SET), live birth rates in fresh and frozen-thawed cycles, and perinatal outcomes in recent years.ResultsSETs and utilization of frozen-thawed embryos increased worldwide over the study period. In 2012 SET utilization in all ART cycles was highest in Japan and Australia/New Zealand (82.6% and 76.3% respectively) and lowest in Latin America (16.0%). While gradual improvements in live birth rates were observed in most regions, some demonstrated declines. By 2012–2013, fresh cycle live birth rates were highest in the U.S. (29%) and lowest in Japan (5%). In Japan, the observed decline in fresh cycle live birth rate coincided with transition to minimal stimulation protocols, transfer of frozen-thawed rather than fresh embryos, and implementation of an SET policy. Similarly, implementation of an SET policy in parts of Canada was followed by a decline in fresh cycle live birth rate. Increasing live birth rates in frozen-thawed embryo cycles, seen all over the world, partially compensated for declines in fresh ART cycles. During 2012–2013 Australia/New Zealand and Japan reported the lowest multiple delivery rates of 5.6 and 4% respectively while the US had the highest of 27%. In recent years, preterm delivery rates in all regions ranged between 9.0 to 16.6% for singletons, 53.9 to 67.3% for twins, and 91.4 to 100% for triplets and higher order multiples. Inconsistencies in the way perinatal outcome data are presented by various registries, made comparison between regions difficult.ConclusionsART practices are characterized by outcome differences between regions. International consensus on the definition of ART success, which accounts for perinatal outcomes, may help to standardize worldwide ART practice and improve outcomes.Trial registrationPROSPERO (CRD42016033011)

Genetics of androgen metabolism in women with infertility and hypoandrogenism

Hypoandrogenism in women with low functional ovarian reserve (LFOR, defined as an abnormally low number of small growing follicles) adversely affects fertility. The androgen precursor dehydroepiandrosterone (DHEA) is increasingly used to supplement treatment protocols in women with LFOR undergoing in vitro fertilization. Due to differences in androgen metabolism, however, responses to DHEA supplementation vary between patients. In addition to overall declines in steroidogenic capacity with advancing age, genetic factors, which result in altered expression or enzymatic function of key steroidogenic proteins or their upstream regulators, might further exacerbate variations in the conversion of DHEA to testosterone. In this Review, we discuss in vitro studies and animal models of polymorphisms and gene mutations that affect the conversion of DHEA to testosterone and attempt to elucidate how these variations affect female hormone profiles. We also discuss treatment options that modulate levels of testosterone by targeting the expression of steroidogenic genes. Common variants in genes encoding DHEA sulphotransferase, aromatase, steroid 5α-reductase, androgen receptor, sex-hormone binding globulin, fragile X mental retardation protein and breast cancer type 1 susceptibility protein have been implicated in androgen metabolism and, therefore, can affect levels of androgens in women. Short of screening for all potential genetic variants, hormonal assessments of patients with low testosterone levels after DHEA supplementation facilitate identification of underlying genetic defects. The genetic predisposition of patients can then be used to design individualized fertility treatments.