Recent advances in in vitro fertilization.

Embryo implantation occurs following complex synchronized physiological and biochemical interactions between the blastocyst and the endometrium 55 . This occurs only if the endometrium is in a receptive state. In humans, an individually defined period called the “window of implantation” (WOI) spans 2–4 days during the mid-luteal phase 56 . Two recent methods used in IVF clinics for the assessment of endometrial receptivity are ultrasound measurement of subendometrial wave frequency and a microarray analysis of putative implantation-associated gene expression, the so-called endometrial receptivity array (ERA). Early studies using transvaginal ultrasound (TVUS) measurement of endometrial thickness hoped to replace invasive techniques like endometrial biopsy for the assessment of endometrial receptivity. A pre-ovulatory endometrium of 7 mm is considered as the cut-off thickness below which suboptimal implantation occurs 57 – 59 . However, pregnancies have been reported in patients with an endometrial thickness as low as 4 mm 60 . A meta-analysis in 2014 concluded that endometrial thickness by itself did not have a high enough positive predictive value for pregnancy 61 . More recent work has focused on TVUS measurement of the subendometrial contractility, observed as “endometrial waves”, that are hormonally responsive in their patterns and propagation directions 62 – 64 . In the follicular phase, these peristaltic waves are directed from the fundus to the cervix (FC waves), but in the late follicular and the ovulatory phase these waves are directed from the cervix to the fundus (CF waves) to assist sperm transportation to the fallopian tube. Following ovulation is a phase of uterine quiescence which would support embryo implantation 65 . During assisted reproductive technology procedures, supraphysiological estradiol levels are generally reflected by increased wave activity. Fanchin et al . observed an inverse correlation between the frequency of subendometrial waves in the luteal phase and pregnancy outcomes 66 . Increasing progesterone exposure was shown to result in diminished wave activity and improved pregnancy rates. Despite these interesting observations, most IVF units at present do not use ultrasound to assess endometrial wave activity. However, in our opinion, this non-invasive tool could be especially valuable in patients with recurrent implantation failure or with inflammatory conditions such as endometriosis to ensure uterine quiescence prior to the embryo transfer. Endometrial histological dating described by Noyes et al . in 1950 67 was considered the gold standard for determining the WOI, thought to be around day 20 to 22 of an idealized 28-day cycle. Endometrial deficiency or “out-of-phase” endometrium is thought to occur in as many as 1 in 4 patients 68 . Multiple randomized studies have cast doubt on the reproducibility of the analysis of endometrial biopsy samples by histologic dating 69 – 71 . A new technique has developed from the transition to microarray molecular analysis using a customized array to identify markers of endometrial receptivity. The ERA based on the analysis of the expression of 238 genes thought to be involved in endometrial implantation may lead to the determination of the personalized WOI 72 . This test is done by obtaining endometrial biopsy samples on day LH surge + 7 in a natural cycle or the 6 th day of progesterone administration during an HRT cycle. Results are expressed as pre-receptive, receptive, or post-receptive. If the result is non-receptive, which happens in 1 in 4 patients 73 , the embryo replacement timing is adjusted, enabling personalized embryo transfer 74 . This technique, although offered commercially, still requires large-scale randomized studies for validation and the results remain controversial.

In this review, we were asked to update the readers of F1000 on the rapidly advancing field of assisted reproduction. There have been too many new technologies that have been developed over the last 5 years to cover in detail in this review, so we decided to focus on advances that have generated some controversy or that are new enough to require further validation in the future. We plan to discuss methods to improve oocyte quality, especially in older women, together with new stimulation protocols that may improve the number of mature oocytes retrieved during an in vitro fertilization (IVF) cycle. We will discuss the present controversy around pre-implantation genetic screening (PGS) and finally focus on some controversial new methods to determine endometrial receptivity. Needless to say, much of what we have to say in this review will reflect our personal opinions in some of these areas.

Recently, a few studies have demonstrated a potential benefit of the use of the oral agent letrozole together with gonadotropin stimulation in IVF cycles, especially in breast cancer patients going through fertility preservation treatment 12 – 15 . The goal of co-administration of letrozole is to reduce serum estrogen concentrations during ovarian stimulation in breast cancer patients. These studies showed that treatment of breast cancer patients with letrozole and gonadotropins throughout the entire stimulation significantly decreased estradiol concentrations as expected but, interestingly, also increased the number of mature oocytes for cryopreservation compared to controls without breast cancer treated with standard COH 15 . As far as we know, only breast cancer patients undergoing IVF treatment have been treated with letrozole during the whole stimulation phase so far. In our opinion, however, this protocol is likely an excellent treatment for normal responders undergoing IVF to lower the dose of gonadotropins required to obtain adequate numbers of oocytes for fertilization and to keep estrogen levels closer to the physiologic range. There are some limited data for the use of letrozole in IVF cycles of normal responders involving co-administration of gonadotropins and letrozole for 5 days in the early follicular phase 16 – 18 . Favorable outcomes related to letrozole were reported, including lower doses of gonadotropin, which decreased the cost of the IVF treatment, and increased numbers of oocytes and mature oocytes while achieving the same pregnancy rate compared to conventional stimulation. More data exist for the use of letrozole in IVF cycles of poor responders. The rationale for co-treatment with letrozole in poor responders is to increase the intrafollicular androgen concentrations, which have been shown to serve as precursors for ovarian estrogen synthesis as well as having a fundamental role in ovarian follicular development by augmentation of FSH receptor expression on granulosa cells 19 . Co-administration of letrozole and gonadotropins has been described to improve the outcomes in poor responders undergoing IVF cycles 20 – 23 . Garcia-Velasco et al . 24 in 2005 evaluated the impact of letrozole as an adjuvant treatment in IVF cycles on intraovarian androgens and cycle outcome. They found that adding 2.5 mg of letrozole for the first 5 days of gonadotropin stimulation significantly increased follicular fluid androstenedione and testosterone concentrations and improved IVF cycle outcome. They found a significantly larger number of retrieved oocytes and a significantly higher implantation rate in the letrozole group compared to the control group. Although the results of these studies are promising, further prospective studies will be needed to confirm the potential benefit of adding letrozole to gonadotropins in both normal and poor responder patients undergoing IVF. Considering the profound effect intraovarian androgens may have on early follicular growth, different protocols have been used to try to increase intrafollicular androgen concentrations in poor responder patients. Pre-treatment with transdermal testosterone was shown to improve ovarian sensitivity to FSH and follicular response to gonadotrophin treatment in low-responder IVF patients 25 and resulted in an increase in the number of cumulus oocyte complexes retrieved as well as improved clinical pregnancy and live-birth rates 26 . Gleicher et al . 27 investigated patients with diminished ovarian reserve who were supplemented with dehydroepiandrosterone (DHEA) for 30–120 days (25 mg 3 times daily). They demonstrated higher AMH levels in the treated patients compared to the non-treated patients, and they also demonstrated improved pregnancy rates. The same group also demonstrated that DHEA may reduce embryo aneuploidy rate 28 and also miscarriage rate 29 . Wiser et al . 30 performed a prospective randomized controlled study of the effect of DHEA supplementation on IVF outcomes among poor-responder patients. He found an improvement in the embryo quality and a significantly higher live-birth rate in the DHEA group compared with controls. The number of eggs and zygotes was similar in both groups. The use of DHEA for older women or poor responders remains controversial because of the paucity of randomized controlled trials.

Elective single embryo transfer has been suggested as the most efficient approach to minimize multiple pregnancies resulting from assisted reproduction treatments, and the traditional morphological evaluation has remained the first-line method for selecting the most developmentally competent embryo from an available cohort. During the last decade, time-lapse imaging (TLI) has emerged as a novel technology that enables continuous evaluation of early embryo development by automated image acquisition every 5–20 minutes and accordingly does not rely on static observations to define a highly dynamic process. Furthermore, it is possible to score embryos without removing them from the incubator, so there is no exposure to changes in light, humidity, temperature, pH, or gas. Many morphokinetic parameters have been identified to correlate with the embryo's ability to create a pregnancy 39 , 40 , and many different embryo-selection algorithms have been proposed to increase the prediction rate. Recently, Barrie et al . 41 performed a retrospective observational analysis that demonstrated a need for the development of in-house embryo-selection algorithms that are specific to the patient, treatment, and environment. Their data suggested that currently available algorithms are not clinically applicable and lose their diagnostic value when externally applied. A recent meta-analysis 42 assessed whether TLI resulted in favorable outcomes for embryo incubation and selection compared with conventional methods in clinical IVF. This analysis included 10 randomized controlled studies and concluded that there is insufficient evidence to support TLI as a superior method compared to conventional methods for human embryo incubation and selection. A well-designed RCT is still needed to evaluate the effectiveness of the clinical use of TLI.

The objectives of PGS for aneuploidy are to select embryos with the highest chance of implantation, to facilitate elective single embryo transfer, and to reduce the risk of chromosomal abnormalities in the offspring. The current embryo biopsy technique for PGS, day 5 multi-cell trophectoderm biopsy, has replaced the old day-3 single blastomere biopsy and is thought to have a much-improved embryo implantation rate. The genetic testing methods for PGS have also achieved higher accuracy with the progression of test platforms from FISH, to aCGH, SNP arrays, Q-PCR, and the current next-generation sequencing (NGS) technology 43 . PGS may demonstrate abnormal results in more than three-quarters of embryos tested. However, recent research showed that some of these diagnosed abnormal embryos may have the potential to develop into healthy babies 44 . Embryo mosaicism and the trophectoderm sampling technique presently utilized are two major reasons for the possible inaccuracy of PGS. Embryo mosaicism is an extremely controversial topic at the moment. Mosaicism of the chromosomal complement in an embryo can be observed at different stages of embryonic development and is thought to arise during mitotic cell divisions after fertilization. Mosaic cells might reside within the inner cell mass, in the trophectoderm, or in both. Also, the distribution of mosaic cells in the blastocyst can be local, patchy, or uniform 45 , 46 . Animal studies found a wide range of mosaic embryos. A recent study using discarded human embryos found similar wide ranges of embryo mosaicism, ranging from 20 to 90% of embryos 47 . Until now, there is not enough clinical data to predict the fate of the embryos with mosaicism, and there is a clinical and ethical debate around whether embryos determined to be chromosomally mosaic could, or should, be transferred. Animal studies have shown that some mosaic embryos can implant and develop into healthy babies through self-correction. A few clinical reports so far have demonstrated normal pregnancies after transferring abnormal PGS embryos. Lledo et al . used aCGH to re-evaluate trophectoderm biopsy samples and found that 13.4% of embryos previously diagnosed as euploid were mosaic 48 . The clinical pregnancy rate was thought to be similar between mosaic and euploid embryo transfers, but the miscarriage rate may be higher in the mosaics. Fragouli, using NGS to analyze trophectoderm biopsies, found a lower pregnancy rate in mosaic embryos compared to euploids 44 . Both studies, however, provided evidence that embryos with certain degrees of mosaicism may develop into healthy babies. Another controversy concerns the accuracy of PGS as it is now practiced. A recent computer modeling study showed that a single trophectoderm biopsy of 5 to 6 cells cannot accurately estimate the degree of embryo mosaicism, casting into doubt the entire value of PGS 49 . Possible ways to solve this problem include multiple trophectoderm biopsies or, better yet, biopsy of the inner cell mass 50 . However, the safety of removing cells from the inner cell mass is not yet established. Assuming an accurate method of detecting mosaic embryos, an ethical controversy is whether the transfer of these embryos would carry a high risk of miscarriage. On the other hand, discarding aneuploid and mosaic embryos using our present biopsy techniques could potentially result in the loss of embryos that have the potential to develop into a normal baby 51 . This area of controversy will not be solved in the near future. An offshoot of PGS and trophectoderm biopsy is the ability to determine the mean mtDNA copy number in the blastocyst. Elevated mtDNA copies at the blastocyst stage have been found to be associated with poorer clinical outcome 52 , 53 . Euploid embryos with relatively low levels of mtDNA at the blastocyst stage were observed to have a higher implantation rate compared to blastocysts with a relatively higher copy number of mtDNA. In keeping with the latter observation, blastocysts from younger patients have been found to have lower average copy numbers of mtDNA compared to older patients 52 , 54 . This difference was evident when all blastocysts were considered together but also when chromosomally normal and abnormal embryos were considered separately. This finding raises the question of whether mitochondria might play a direct role in the decline of female fertility with age. Fragouli et al . 54 examined mtDNA quantity in relation to chromosome status. They found that chromosomally abnormal blastocysts tended to contain significantly larger amounts of mtDNA compared to those which were characterized as being euploid. The elevated mtDNA levels in the abnormal and the older embryos might be a consequence of a compensatory mechanism aimed to increase the ATP generation of compromised mitochondria of reduced function. At present, the analysis of mtDNA copy number at the blastocyst stage as a way to improve live-birth rates is controversial, and further research is ongoing.