The production of ATP via OXPHOS involves a complicated process including 5 complexes located on the inner mitochondrial membrane ¹ . Ubiquinone or coenzyme Q10 (CoQ10) plays an important role in this process, as it has antioxidant properties, controls cellular redox, and affects various signaling pathways ^{5, 6} . The concentration of CoQ10 in most tissues decreases after 30 years of age in humans ^{7, 8} , and this decline in CoQ10 may contribute to the aging process, since it coincides with the decline in fertility and increased rate of aneuploidies. Ben Meir et al. in 2015 showed that supplementation of CoQ10 in an aged animal model delayed decline in ovarian reserve, restored oocyte mitochondrial gene expression, and improved mitochondrial activity with a significant reduction in oocyte aneuploidy. As a result, these aged mice had more oocytes with stimulation, the developmental potential of the oocytes was improved, and more offspring were born compared to old animals receiving placebo ¹ . As a follow-up study, conditional disruption of the gene PDSS-2, resulting in isolated CoQ deficiency in the oocytes of young animals, resulted in phenotypic changes characteristic of oocyte mitochondrial dysfunction associated with aging ¹ . By feeding the animals CoQ10, these changes could be reversed.

Since CoQ10 administration in old animals was shown to have beneficial effects on reproductive outcomes, one could speculate that older women might have the same benefits when supplemented with CoQ10 ¹ . At present, there is much research activity in this area. Although the animal model appears promising, the aging process differs greatly between mice and women because of the huge difference in lifespan. CoQ10 administration for 12–16 weeks in mice is likely equivalent to years of use in humans ¹ and thus additional large-scale clinical studies are needed.

Other attempts to overcome ooplasmic aging have utilized oocyte manipulation at a subcellular level. Cohen et al. performed ooplasmic transfer from donor oocytes into mature oocytes of patients who had failed multiple IVF cycles because of poor embryo development and showed healthy live births as a result ⁹ . Since cytoplasm contains mitochondria, mitochondria from the donor ooplasm were also transferred into the recipient eggs and were believed to be the most important mediator of improved embryogenesis. Several of the healthy babies tested were found to be heteroplasmic, i.e. to have mitochondrial DNA (mtDNA) derived from the mother and the cytoplasm donor ¹⁰ , and the procedure of ooplasm donation is no longer used. However, autologous mitochondrial transfer has now extended this previous work with ooplasm transfer. In this new technology, mitochondria similar to oocyte mitochondria are isolated from oocyte precursor cells in the superficial epithelial layer of the patient’s ovary and are injected into the patient’s own oocytes at the time of fertilization. This mitochondrial injection has been demonstrated to improve embryo development and lead to live births in women with previous poor embryo development ¹¹ . This technique, and even the presence of oocyte precursor cells, remains controversial and needs proper randomized control studies for validation.

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.

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.

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.