UI can occur, even in the presence of regular menstrual cycles, as a result of diminished ovarian reserve, defined as a reduced quantity and quality of the remaining population of primordial follicles within the ovary than would be expected for a given chronological age . Even though chronological age is the most important determinant of ovarian reserve, evidence has shown great variability in the rate of ovarian ageing [17]. Therefore, women with prematurely ageing ovaries (PAO) [18] may be mistakenly diagnosed with UI, since nobody would expect such fertility decline be based on their age alone.

In this setting, female fertility loss may be assessed by tests of ovarian reserve, such as day 3 serum follicle stimulating hormone (FSH) , anti-Müllerian hormone (AMH) levels, and antral follicle count (AFC) [19]. FSH and AMH do not measure the same ovarian reserve parameters. Indeed, FSH is mostly representative of the last 2 weeks of follicular maturation when follicles come to be gonadotropin-sensitive [20], while AMH, exclusively produced by the granulosa cells of early antral and preantral follicles, mainly reflects the earlier stages of folliculogenesis [21, 22, 23]. AFC, visualized by transvaginal ultrasound, is considered the best predictor of ovarian response to stimulation [24], since it correlates with the number of oocytes retrieved and ART outcomes in terms of ongoing pregnancy [25, 26].

In this context, ovarian reserve testing and genetic screening could be of great importance, especially in women < 35 years old, to reveal cytogenetic abnormalities, as in the case of the Fragile X syndrome (FXS), that is caused by an increased number of trinucleotide (CGG) repeats on the fragile X (FMR1) gene and linked to premature ovarian failure [27].

Such genetic and ovarian reserve screening may help women potentially have some knowledge about the duration of their own reproductive window and help clinicians counsel patients and direct them to an appropriate treatment option, which in some cases may include gamete donation [19].

Several abnormalities of ovulation can occur at the gonadal level, such as rupture of the follicle without release of the oocyte, maturation of an empty follicle that does not contain an oocyte, ovulation with inadequate luteinization, incomplete maturation of a follicle resulting in atresia, and finally luteinization of a follicle, under the action of luteinizing hormone (LH) , without its rupture and with entrapment of the oocyte, which is also called the luteinized unruptured follicle (LUF) syndrome [28]. This ovulatory dysfunction is considered as a potential cause of female UI [29, 30] and has been linked to endometriosis and pelvic adhesions [31]. Many publications have appeared since 1978 to describe this syndrome, but the exact mechanism by which the ovulatory follicle fails to rupture is not clearly known. Different mechanisms have been suggested for this syndrome, such as a chronic follicular inflammatory-like reaction involving inhibition of synthesis of prostaglandins [32], luteal phase defect [33] or a primary granulosa cell defect [34] .

LUF is characterized by normal endocrine signs of ovulation, such as secretory endometrium, normal production of progesterone, and duration of the luteal phase [20], and is usually diagnosed by ultrasound demonstration of a follicle that does not change in size or consistency after ovulation should have occurred [35]. LUF has been demonstrated in both spontaneous and stimulated cycles [36] and it is estimated to be present in 6–12 % of cases of female subfertility and in 20–25 % of cases when ovarian stimulation is used [37], but the incidence varies depending on the methods of diagnosis such as LPS or ultrasound or steroid hormone concentrations in peritoneal fluid [38]. Qublan et al. found the recurrence rate of LUF increased from 25 % in the first cycle of intrauterine insemination (IU) to 78 and 90 % in the second and third cycle, respectively [25]. These data are consistent with those reported by others [23–26], but in contrast to previous studies in which LUF was associated with no recurrence rate in subsequent cycles [39, 40] .

It has been shown that mild endometriosis can affect normal tubal function, as well as other reproductive processes and since this diagnosis is clinically frequently missed, it may represent a possible etiology of UI. For this reason, diagnostic LPS could be considered as an integral part of the evaluation, and the role of tubal function, not just patency should not be underestimated [41].

The prevalence of endometriosis among women who underwent LPS for an infertility evaluation has been reported to be in the range of 10 [10]–50 % [42]. Even if performed by experienced laparoscopists, the diagnosis of subtle endometriosis can be hard to make because the disease is often microscopic and presents with atypical lesions [43, 44], and as such may be underdiagnosed.

Evidence shows that endometriosis may also affect IVF outcomes [45, 46] interfering with many aspects of the process, including follicular development, oocyte retrieval, fertilization and embryo development [47, 48], distortion of adnexal anatomy and creating an adverse peritoneal environment characterized by increased inflammatory cytokines, oxidative stress [22, 49, 50, 51], and augmented number of peritoneal fluid macrophages [52–54]. If endometriosis does so in vitro, it can be expected to have similar effects in vivo leading to impaired conception [5]. Studies have demonstrated that, even in mild cases of endometriosis, pregnancy outcomes may have been affected by subtle tubal abnormalities [55, 56], thus reflecting microscopic endometriosis in the fallopian tubes, which can never be totally excluded, even by LPS [19–20]. Furthermore, data on patients undergoing LPS for infertility indicate that of those who have no macroscopic endometriosis at LPS, at least 6 % have microscopic lesions [57, 58] and thus confirms how this diagnosis , especially in patients with infertility may be underestimated [59].

The fallopian tubes play an important role in sperm transport , oocyte capture and transport, fertilization, and early embryo development [60]. Abnormalities in any one of these functions cause defective transport of the oocyte and impaired fertilization, through alterations in tubal peristaltic or ciliary activity [61], which may affect one or both fallopian tubes.

Tubal function can be abnormal despite documentation of tubal patency [62, 63], but HSG has limited value in evaluating tubal function and peritubal disease [64, 65] and may be less accurate in detecting tubal disease than LPS [66, 67]. Moreover, a study performed in an infertile population showed that HSG missed at least one tubal abnormality in 84 % of the cases [68]. Despite all these limitations, HSG represents the first line tool to evaluate tubal status, because of its safety and low cost. LPS remains the gold standard for the evaluation of mechanical factors affecting the fallopian tubes , but it can miss proximal disease [65] and cannot be used to directly observe the ampulla, where the fertilization between oocyte and sperm occurs. This may be explored by salpingoscopy . Some researchers think that salpingoscopy could be informative in patients with UI, since it can identify nonobstructive tubal diseases such as fibrosis, adhesions, debris, and foreign bodies [69].

Furthermore, the impact of chlamydial infection in the etiology of tubal pathology secondary to salpingitis [70–72] on female fertility is well documented. Although neither HSG or LPS may identify tubal pathology secondary to chlamydial infections in the absence of overt occlusion or peritubal adhesions, subfertile women with a positive Chlamydia trachomatis antibody have lower chances for pregnancy than seronegative women [73]. This evidence confirms the validity of assessment for chlamydial serology in women under fertility investigation [74, 75].

A decrease in fertilization has been documented in IVF cycles performed in couple with UI, suggesting gamete defects as potential causes of UI.

Absence of fertilization has been shown in a prospective study from Ruiz et al. [76], in couples with UI and mild endometriosis undergoing IVF/ICSI after four failed intrauterine insemination cycles. It has been found that 11.4 % of these couples suffered fertilization failure with standard IVF, and if ICSI would not have been employed, they might have had no embryos available for transfer. In this study, ICSI did not increase fertilization rates over standard IVF in case of UI, but avoided complete fertilization failure in those patients.

Alboughar et al. [77] reached similar conclusions with regards to gamete dysfunction in couples with UI, in which the rate of fertilization failure was 22.7 %, very similar to those observed in other studies in couples with UI [78, 79].

The use of assisted reproductive techniques has been important not only for therapeutic reasons, but also because it helps to understand the complex process that leads to conception in a given couple. Indeed, it is noteworthy that there is a high incidence of chromosomal abnormalities in human embryos cultured in vitro [80] and that many repetitive implantation failure (RIF) cases are due to embryonic defects, including chromosomal aneuploidy , which increase with maternal age [81] and with the number of previous failed IVF cycles [82, 83]. Different therapeutic options have been proposed to improve the outcome of these patients, including assisted zona hatching [84] and coculturing embryos to the blastocyst stage [85]. However, other studies have found dramatic declines in implantation and pregnancy rates using blastocyst culture in RIF patients due to the limited development of these embryos in extended culture [86].

In a randomized controlled trial of infertile couples with RIF and prior transfers of good-quality embryos, Rubio et al. reported a trend towards an increased incidence of genetic abnormalities in embryos from these couples and an improvement in live-birth rates per transfer by selection of the healthiest embryos with PGS, highlighting the mechanisms of action by which chromosomal abnormalities can have an impact on UI.

An altered endometrial receptivity may interfere with apposition, adhesion, or penetration of the embryo and results in a failed implantation [87] .

Since the 1950s, traditional histologic evaluation of the endometrium performed by pathologists, has been used as a predictor of endometrial receptivity [88, 89], the clinical relevance and reproducibility of which has been questioned in randomized studies [90, 91]. The development of microarray technology [92] helped to analyze the expression of thousands of genes at the same time in an endometrial sample of development in the peri-implantation period. On those grounds, and consistent with the findings that endometrial receptivity may be related to its transcriptomic profile, molecular assessment of endometrial receptivity has been developed [93, 94], a molecular diagnostic tool that contains 238 expressed genes coupled to a computational predictor, which is able to identify endometrial samples within the window of implantation , independent of their histological appearance. The endometrial receptivity array (ERA) test may help to identify patients with implantation failure caused by a nonreceptive endometrium, improving the ability to control the endometrial environment for implantation. Moreover, an endometrial database (EDB) (http://​www.​endometrialdatab​ase.​com) has been created to facilitate the exchange of information on the genomics of endometrial receptivity, for the improvement of knowledge in this field worldwide [95] .

UI may reflect a malfunction of the endometrial-embryo “dialogue” in the early phases of implantation that leads to early pregnancy loss (EPL) , or biochemical pregnancy (BP) which could be erroneously interpreted as a failure to conceive. EPL or BP are defined as increases in beta-human chorionic gonadotropin (B-hCG) at the end of the luteal phase due to embryonic implantation that does not result in a clinical pregnancy. The development of sensitive immunoassays for the detection of urinary B-hCG has allowed for detection of a pregnancy within a few days of embryo implantation, which shows high rates of BP in spontaneous conception [96]. Moreover, data from patients who have undergone ART and PGS show that many preclinical implantation failures are due to chromosomal alterations and are found in high rates in natural (25 %) [97] and ART conceptions (40 %) [98]. Chromosomal aberrations are probably not the only cause of EPL. Indeed, an altered endometrial receptivity due to environmental factors, such as age and excessive ovarian stimulation in IVF cycles, may also play an important role in the etiologies of this disorder, as shown by Troncoso et al. [99] .

There is clear evidence that sperm transport through the female genital tract from the cervix into the tubes, assisted by cervico-fundal uterine peristaltic contractions [100, 101], is altered in patients with UI and endometriosis which results in impaired uterine contractility, documented by hysterosalpingoscintigraphy (HSSG) [102]. Data have shown that endometriosis is associated with uterine hyperperistalsis and dysperistalsis , which may cause impaired or total failure in sperm transport capacity respectively, especially when diffuse adenomyosis is also detected [103]. Dysperistalsis is associated with reduced natural conception rates [104, 105] and consequently IVF/ICSI may be required even in couples with otherwise patent fallopian tubes and normal semen parameters.

Infertility is unexplained after thorough evaluation in about 15–30 % of cases and constitutes a multifactorial disorder of reproduction, as discussed in this chapter. Many potential etiologies of UI have been proposed here, including ovarian factors, fertilization failure , failure of the embryo to develop, failure of implantation and impaired or total failure in oocyte and sperm transport due to altered tubal and uterine function .

Increasingly, complex ART options have led to the finding of many causes of infertility that in the past have only been suspected, but can now be diagnosed, as in the case of chromosomal aneuploidies identified with PGS. However, multiple potential etiologies of UI could coexist with identified causes for infertility and therefore many couples with identified factors may fail to conceive despite receiving appropriate treatment for the identified causes. It is, therefore, imperative to perform a complete and thorough evaluation of the infertile couple, including evaluation for these subtle etiologies, even in couples whose infertility evaluation has revealed a potential etiology.