Unexplained Infertility:Pathophysiology, Evaluation and Treatment
Abstract
A unique compilation of the experience and insights of leading specialists, clinicians and scientists with expertise in male and female infertility ▶ An invaluable tool for basic scientists with an interest in reproductive medicine and for clinicians working in the field of infertility-urologists, gynecologists, reproductive endocrinologists and embryologists ▶ Practically organized into sections on epidemiology, male and female pathophysiology, evaluation, medical and surgical management strategies and ART Written and edited by leading, internationally recognized clinicians and scientists in reproductive medicine and related fields, this unique text is a practical and comprehensive review of the clinical and scientific significance of unexplained male and female infertility and its management. The book is divided into thematic sections to ensure the most useful presentation of topics, opening with definitions and epidemiology of unexplained infertility, including discussion of the WHO's cutoff values for human semen characteristics and its ramifications. Sections covering male and female reproductive pathophysiology follow respectively, covering biological, genetic and environmental causative factors, with a subsequent section on evaluative techniques for male and female patients. Expectant, medical and surgical treatment strategies comprise the fifth section of the book, where active interventions and outcomes of each treatment modality are carefully considered. The final section discusses assisted reproductive techniques to manage unexplained infertility, such as intrauterine insemination and in vitro fertilization, as well as future perspectives. Thoughtful and enlightening, Unexplained Infertility: Pathophysiology, Evaluation and Treatment will be an invaluable resource for all clinicians and scientists working in the fields of reproductive medicine and infertility.
Chapters (32)
Infertility is customarily defined as failure of a couple to conceive after 12 months of unprotected regular intercourse. However, after extensive evaluation of both partners, 20-30 % of infertile couples remain childless without identifiable causes, according to the routinely used tests, and such a category is known as unexplained infertility (UI). Potential etiologies of UI encompass couple’s miscomprehension of the concept of the female fertile window, improper coital techniques, erectile dysfunction, as well as molecular and functional causes of male and female infertility. Contemporary advanced technologies have demonstrated various ultrastructural, molecular, and genetic etiologies in male or female partners with UI. This book is aimed to analyze the full spectrum of UI problems from the presentation of the very basic science studies of male and female reproductive system to the detailed examination of the potential male and female causes of UI. Further insight is contemplated into the treatment options ranging from expectant management to active interventions.
Male factor is a common cause of infertility among couples trying to achieve a successful pregnancy. Half these patients have no identifiable cause for their infertility. Patients are classified as having unexplained male infertility (UMI) when there is no abnormality on history and physical examination, semen analysis, and hormone evaluation. The prevalence of UMI is approximately 15 % (range 6-37 %) in the general population. There are multiple immune, genetic, and environmental factors that may contribute to this condition. The objective of this chapter is to highlight the epidemiology and terms used to define unexplained male infertility.
![Table 3 .1 Cutoff reference values for semen characteristics as published in consecutive WHO manuals. (Esteves et al. [6], with permission from Excerpta Medica, Inc.)](https://www.researchgate.net/profile/Sandro-Esteves/publication/283535420/figure/tbl1/AS:667815380004867@1536231002581/Cutoff-reference-values-for-semen-characteristics-as-published-in-consecutive-WHO_Q320.jpg)
In 2010, the World Health Organization (WHO) published new reference criteria for human semen characteristics. Important methodological issues, including patient selection and methods for semen assessments, elucidate why reference values are markedly lower than those earlier reported. The validity of the new criteria as a representation of the semen quality of fertile men has been questioned as they have been generated from a small group of recent fathers of restricted geographic regions. The 2010 WHO criteria are likely to have a significant clinical effect as more men will be classified as having unexplained male infertility. This may lead to a deferment of referral and could delay the definitive diagnosis and management of the infertile couple. Due to the several inherent limitations of semen analysis as a surrogate marker of male infertility, physicians should exercise caution when interpreting results. Semen analysis alone is usually insufficient for the diagnosis, as it does not account for sperm dysfunction, such as immature chromatin, oxidative stress (OS), and DNA damage. A male infertility evaluation must go far beyond a simple semen analysis, as it has to be complemented with a proper physical examination, a comprehensive history taking, and relevant endocrine, genetic, and other investigations.
There are inherent difficulties in defining infertility and unexplained infertility (UI), and the scientific community is yet to reach a consensus opinion. These difficulties stem from the differences in the clinical, epidemiological and demographic approach in defining infertility.
Various definitions for infertility have been proposed. The most up-to-date definition states that infertility is a disease, defined by the failure to achieve a successful pregnancy after 12 months or more of appropriate, timed unprotected intercourse or therapeutic donor insemination. UI itself is a diagnosis of exclusion made in the presence of a normal semen analysis when tubal patency and normal ovulatory function are established. It can be further classified into male and female UI, a concept that has only recently emerged.
Unexplained female infertility could potentially be attributed to several causes and this association is directly related to the extent that the female is investigated.
Studying the epidemiology of infertility and UI is complicated by the disparity in defining the disease as well as the heterogeneity of the studies. It is estimated that in about 30 % of infertile couples seeking help of a reproductive specialists, the cause is unexplained. The prevalence of UI around the world does not vary significantly but the aetiologies do.
Investigating and treating women who suffer from UI is constantly evolving in light of new evidence and technologic advances that allow us to identify potential causes that were previously elusive. Therefore, the definition of the disease and its epidemiology are dynamic and may change in the future.
Spermatogenesis is a sequence of highly intricate stages by which an undifferentiated diploid spermatogonium matures into a specialized, genetically unique haploid spermatozoon. Within the Sertoli cells, both mitosis and meiosis are responsible for transforming the diploid spermatogonial cells into unique haploid spermatids. This process requires the assistance of hormones regulated via the hypothalamus-pituitary-gonadal axis-namely, gonadotropin-releasing hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). However, not all spermatogonia are destined to mature. In fact, most undergo apoptosis and are phagocytosed. Through spermiogenesis, spermatids elongate to form spermatozoa, which then leave the Sertoli cells and enter the epididymis for final maturation. Here, they acquire motility and acrosomal function, which are necessary for successful fertilization. This entire process from production to ejaculation of mature spermatozoa takes, on average, 64 days to complete. Essentially, spermatogenesis and spermiogenesis create fully functional spermatozoa that can travel efficiently through the female reproductive tract to the ovum and allows for the contribution of exclusive male genes to the offspring genome. This chapter serves as a comprehensive overview of sperm biology from production to ejaculation.
Fertilization is undoubtedly one of the most complex and tightly regulated biological events in life. Understanding both the cellular and molecular mechanisms that underlie successful fertilization has great implications toward the etiology of human infertility, and grasping the role and function of spermatozoa in this regard may present a potential in the field of idiopathic male infertility.
Spermatozoa are deposited in the female reproductive tract during intercourse, where they have to gain both motility and fertilizing ability in a foreign and hostile environment. The spermatozoon’s ability to fertilize an oocyte is gained through a series of events, including capacitation and the acrosome reaction, which are initiated at very strategically specific points in the female reproductive tract, to ensure that fertilizing capability is maintained until the oocyte can be reached. The journey to the oocyte is an arduous one, and the spermatozoa must complete it independently and with very limited resources, as they are not directly connected to the blood supply of the female.
In the female reproductive tract, spermatozoa encounter obstacles at practically every point they reach. However, evolution has provided the physiologically superior spermatozoa with the means to overcome these challenges. In the vagina, spermatozoa must face gravity, an acidic environment, as well as an influx of female immune cells, all of which is overcome by the coagulation of the seminal plasma in the anterior vaginal regions close to the cervical opening. Entrance to the cervix can only be gained around the time of ovulation, but once inside, the mature and vigorously motile spermatozoa are favored and protected from microbes in the deep cervical channels, as they make their way to the uterine cavity. In the uterus, spermatozoa are swept toward the fallopian tubes mainly by smooth muscle contractions of the uterine wall. The fallopian tubes provide a safe haven for spermatozoa, due to the absence of female immune cells and the existence of a functional sperm reservoir intended to maintain spermatozoa in a fertile state until the oocyte can be reached and fertilized.
Male factor infertility contributes to one-half of all infertile couples. Of these, approximately 40 % have an unknown cause. This subgroup can be divided into separate categories based on the results of two properly obtained semen analyses: idiopathic male infertility (IMI) and unexplained male infertility (UMI). While both groups exhibit normal physical exam findings and endocrine evaluations, they differ in that IMI have abnormal semen analyses, while UMI have normal semen parameters. This chapter will highlight the potential etiologies of UMI.
Approximately 12 % of the subfertile males attending our tertiary center for male reproduction, in which all male partners undergo andrological examination regardless of semen analyses results, are classified as having infertility of unknown origin. In several of these cases, etiologies may be is gene related including chromosomal defects, gene mutations, DNA damage, sperm chromosomal abnormalities, and epigenetic disorders. In this chapter, we review the basic genetic concepts to provide an overview of relevant points to be considered when assessing genetic causes of unexplained male infertility. In addition, we present the genetic conditions that may impair fertility without affecting semen analysis results, and the diagnostic methods to identify these disorders. Finally, we propose a genetic workup plan to better evaluate and counsel males with unexplained infertility (UI), and therefore provide an oriented therapeutic choice.
The immune system plays an important role in male infertility. The testis is an immune-privileged site. Sperm cell has several "nonself" antigens that develop during later stages of spermatogenesis. These "nonself" antigens are sequestered from the immune attack by the blood-testis barrier. The most frequent phenomenon of immune pathology in infertility is the development of antibodies directed to these "nonself" antigens that are relevant to sperm motility, function, fertilization, and fertility. There are several etiological factors and sites in the male genital tract that can induce antibodies to sperm. The antisperm antibodies (ASAs) can be detected by various methods, including the mixed antiglobulin reaction (MAR) test, immunobead binding test (IBT), and enzyme-linked immunosorbent assay (ELISA). The presence of ASAs has been shown to reduce fertilization rates in the in-vitro fertilization (IVF) procedure used for the treatment of male infertility. For the treatment of immunoinfertility, the suppression of antibody production using steroids (glucocorticoids) has not yielded successful results. Several in-vitro methods have been used to remove antibodies bound to sperm for treatment. Intracytoplasmic sperm injection (ICSI) technique has been successfully used to bypass sperm antibodies that are relevant to fertilization. However, the embryos may degenerate even after successful fertilization. In conclusion, the available data indicate that immunological factors, including ASAs, play an important role in pathogenesis of unexplained male infertility. As additional fertility-related sperm antigens and mechanisms become delineated, a larger subgroup of "unexplained infertility" is becoming a part of "immunoinfertility." © Springer Science+Business Media, LLC 2015.
The aim of this chapter is to clarify the concept of unexplained male infertility (UMI). In this chapter we will discuss the difference between UMI and idiopathic infertility, the components of an infertility workup, and the parameters addressed in a routine semen analysis necessary to diagnose unexplained infertility. Furthermore, it elaborates upon the various etiologies suggested to play a role in UMI, such as coital problems, physical aberrations within the male reproductive system, compromised genetic integrity, immunologic dysfunction, and fertilization defects. Most importantly, this chapter emphasizes the implications of oxidative stress in the pathogenesis of unexplained infertility as well as how these oxidative stress-induced changes can be measured. Treatment options for those with UMI are also discussed.
It is estimated that about 20 % of couples with difficulties to conceive have no identifiable infertility conditions based on the routine investigation. Such couples are categorized as having unexplained infertility (UI). Concerning the male partner, results of semen analyses are usually taken as surrogates of the fertility potential. Semen analysis, however, must go far beyond counting spermatozoa and assessing motility and morphology, as routinely performed. After ruling out female infertility factors, specialized andrology testing may assist in unraveling dysfunctions related to immunologic disorders, excessive oxidative stress, and other problems affecting spermatozoa at the cellular and subcellular levels. Recent advances in the field of genetics, proteomics, and metabolomics hold promise and some tests, including those that measure sperm chromatin integrity and reactive oxygen species (ROS), have already been added to the laboratory male infertility investigation. This chapter focuses on the traditional and novel clinically available laboratory methodologies for the investigation of the male partner of couples with UI, including antisperm antibodies (ASA), sperm fertilization defects, levels of ROS both in the sperm and seminal plasma, and sperm chromatin integrity. The identification of dysfunctions by cost-effective and accurate specialized sperm function tests helps not only to identify the cause of male infertility but also to define treatment strategies.
Seminal quality has deteriorated rapidly over the past 50 years, making it an increasingly prevalent and relevant issue in unexplained male infertility (UMI). Researchers believe that the ever-changing environmental and lifestyle conditions to which the human body is exposed throughout an entire lifespan contribute greatly to this deterioration. Developments in industry and changes in modern lifestyle give rise to a range of different factors such as exposure to chemicals and toxins, harmful environmental agents and adverse lifestyle factors, all of which the body and consequently the reproductive system, has to cope with. Environmental insults during any phase (gestational, childhood and adulthood) of human development can mediate mechanisms disturbing the morphologic, hormonal, or oxidative aspects of testicular tissue and can have severe and irreversible effects on spermatogenesis in a subject or its offspring.
This chapter aims to address some of the most prominent toxins and environmental factors affecting male infertility today, briefly discussing some of the possible effects that these toxins may have as the result of exposure of the male reproductive system to these adverse elements.
Although the analyses of the results of evaluating the effects of environmental factors on the male reproductive system are not without their constraints, this chapter certainly provides enough compelling evidence to conclude that environmental and lifestyle factors play at least some role, if not a definitive one, in the development of UMI.
Unexplained infertility (UI) indicates the absence of an apparent cause for the failure of a couple to achieve pregnancy after 12 months of unprotected intercourse. UI is a multifactorial disorder of reproduction that occurs in approximately 15-30 % of couples presenting with infertility. Despite significant improvements that have occurred in the diagnosis and treatment of reproductive disorders and therefore many potential etiologies of UI have been better understood, many couples still have no explanation for their infertility, even after the most accurate diagnostic workup. In this chapter, we discuss the combination of female factors that potentially underlie UI such as ovarian factors (including abnormal ovarian folliculogenesis and ovulatory dysfunction), tuboperitoneal factors, fertilization failure, alterations in endometrial receptivity and embryonic factors. UI should not be regarded as a permanent condition but rather a relative failure to achieve pregnancy, and as such, it should be better considered as subfertility, since time may lead these couples to conceive without treatment, with the duration of infertility and the female age being the most important prognostic factors. Nevertheless, treatment-independent live-birth rates have been estimated to be between 40 and 60 % at 3 years, the results of treatment outcomes for UI are promising.
Oxidative stress is an imbalance between free radicals and antioxidants. It has been demonstrated that oxidative stress may play an important role in subfertility. Studies have shown a close relationship between oxidative stress and polycystic ovarian syndrome and endometriosis. However, the relationship between oxidative stress and unexplained female infertility has not been studied in detail. This chapter reviews the possible associations between oxidative stress and the underlying mechanisms that contribute to unexplained infertility. The correlations between oxidative stress and oocytes, folliculogenesis, ovulation, fertilization, implantation, oocyte and embryo quality are also discussed. Articles were identified through a search of the Pubmed database. It has been found that oxidative stress plays an important role in etiologic factors that may contribute to unexplained infertility such as defective endometrial receptivity, impaired oocyte quality, premature ovarian failure, minimal and mild endometriosis, tubal disease, pelvic adhesions, and immunological and endocrinological abnormalities. Moreover, it has been demonstrated that reactive oxygen species can also negatively affect ovulation, fertilization, implantation, embryo quality, and pregnancy rates. However, the use of antioxidant therapy in that group remains unclear. Further studies are needed to show the effectiveness of antioxidants in that group.
There is increasing concern about the recognized and potential effects of environmental contaminants on reproductive health. While there are limited clinical data regarding most chemical exposures and human reproduction, studies in laboratory animal models and wildlife underscore the vulnerability of the female reproductive system to many environmental insults during various windows of development and across the life cycle. Herein, we review published data implicating select environmental contaminants that may compromise reproductive capacity in humans as well as supporting data from in vivo and in vitro animal studies. More specifically, we discuss the realized and hypothesized impacts of exposure to cigarette smoke, polychlorinated biphenyls, dioxins, polybrominated diphenyl ethers, perfluorinated compounds, and widespread toxic elements on female reproductive function with a focus on their potential role in unexplained female infertility. The difficulty of studying widespread low-level exposure to reproductive toxicants in human populations introduces a substantial challenge into establishing causal relationships, and epidemiologic data alone are inconclusive. Yet a “weight of evidence” strategy, incorporating the results of experimental studies in vivo and in vitro as well as human studies suggest environmental pollutants play an important role in unexplained female infertility.
A substantial diminution in sperm number occurs as they transverse the cervix. From an average of 200-300 million sperm deposited in the vagina, only a few hundred achieve proximity to the oocyte. Given this expected high spermatozoa loss, a slight modification in cervical mucus may rapidly transform the cervix into a "hostile" environment, which, together with changes in vaginal environment and cervix structure, may prevent spontaneous conception and be a cause of infertility. The cervix generally functions as an effective barrier to sperm and the production of mucus is essential to transport them from the vagina to the uterine cavity. In this chapter, we explain the physiological role of the vaginal pH and cervical mucus in fertility, and describe several conditions that can render the cervical mucus hostile to sperm and, therefore, be implicated in the pathophysiology of unexplained infertility.
Unexplained infertility is a diagnosis of exclusion after the standard tests for ovulation, tubal patency, and sperm have been performed. Some of the patients with unexplained infertility may have abnormal endometrial receptivity. Human implantation is a group of processes which is complex and is far from being completely understood. During the time of implantation not only structural but molecular changes also occur. The "window of implantation" (WOI) is defined as the phase in which the endometrium is receptive to the embryo. The first attempt to evaluate the endometrium was with conventional histology and specific changes were noted based on days of progesterone exposure. However, this is not an accurate or reliable evaluation of the endometrium. Ultrasound evaluation of the endometrium can provide limited value in the study of endometrial receptivity regarding uterine contractility and perfusion. Multiple advances to identify molecular markers have been achieved although it is unlikely that a single molecule can be used in the clinical setting due to the inherent complexity and overlap of molecules and receptors related to implantation. A test of endometrial receptivity using genomic technology appears promising as a customized microarray with selected genes that have been described to be important during the WOI is able to identify endometrial samples that are thought to be "receptive" regardless of their histology.
Unexplained infertility is a “waste basket” diagnosis, encompassing about half of couples experiencing infertility. That infertility is unexplained in at least 50 % of our patients should serve as evidence that we have failed to dissect out the different etiologies comprising this heterogeneous group, while at the same time advancing assisted reproductive technologies. So, while 50 % or more of infertile couples remain undiagnosed, the great majority of them will conceive. And the question, given this reality, is: how much does an accurate diagnosis matter if we can help such couples achieve a successful pregnancy?
Unexplained infertility (UI) is a diagnosis of exclusion for infertile couples; within an otherwise asymptomatic patient with a normal clinical examination and ultrasound evaluation revealing no pelvic pathology, UI may be associated with undiagnosed pelvic diseases. We may speculate whether UI is really unexplained or if there are undocumented subtle pathological pelvic conditions that might interfere with the processes involved in natural fertilization. Subtle endometriosis may be related to various mechanisms involved in the different stages of fertilization, embryo development, and implantation, and there is speculation about its role in cases of UI. There is no consensus about whether milder stages of endometriosis are a cause of infertility or merely an incidental finding in some patients with UI. Studies by our group and others have shown that even cases of milder disease may inhibit mechanisms involved in conception, such as follicular steroidogenesis and oocyte competence. There appears to be an increase in oxidative stress (OS) markers in both the follicular and peritoneal compartments of patients with endometriosis as well as a possible increase in enzyme expression in cumulus granulosa cells (CC) which may alter oocyte development.
Cases of UI, especially those experiencing a long duration of infertility, may be due to subtle endometriosis with possible alterations in the mechanisms of oocyte maturation and response, fertilization, as well as later embryonic implantation. Assisted reproduction is an effective treatment option that can overcome as of yet unidentified intrinsic disturbances in patients with subtle endometriosis and UI.
The diagnosis of unexplained infertility should not be made without diagnostic laparoscopy until sub-optimal ovulation, inadequate endometrial development, cervical factor, and medical disorders such as hypo or hyper thyroidism and insulin resistance of Type II diabetes have been eliminated as causes. Studies that report more than 10 % of infertility as unexplained omit one or more of these tests. When these potentially correctable factors have been ruled out there will remain a small percent of couples whose infertility can be explained when in vitro fertilization (IVF) is performed. The findings in these cases are often either oocytes of poor quality, or failure of fertilization and embryos that cease to develop or fail to implant. Results of preimplantation genetic screening (PGS), in IVF cycles performed for age and unexplained infertility, suggest that many cases of infertility are due to aged or defective gametes. However, proceeding directly to ovulation induction (OI) and IVF without diagnosing and treating an underlying medical cause may impair the patient’s future health and fetal development.
Infertility affects approximately 15 % of couples desiring conception and male infertility underlies nearly half of the cases. However, despite the diagnostic advances in male infertility field, approximately 30 % of patients still have no apparent cause for their infertility problem. Although it is anticipated that future developments will allow identification of the etiology for subfertility in these patients, at present they are considered unexplained disorders that challenge specific treatment recommendations. The use of assisted reproductive techniques (ART) to overcome male infertility is increasing, and due to its effectiveness, this treatment modality has been suggested by some to be used as the treatment of choice in the majority of the cases of male factor infertility regardless of etiology. Although the use of ART may allow infertile couples to achieve pregnancy rapidly, the fear of transferring the unnecessary burden of invasive treatment on healthy female partners weigh down this treatment option heavily. The true litmus test for male fertility remains the ability to generate pregnancy in vivo. Although seminal parameters are used as a surrogate measure of a man’s fertility potential, it is definitely not a direct measure by any means. Seminal parameters between the reference ranges not reflect defects in sperm function. Thus, the assessment of sperm structure and function has gained interest and resulted in the development of the new techniques that are presented in this chapter.
In couples with unexplained infertility, no convincing evidence is available that treatment with intra uterine insemination (IUI) with or without ovarian stimulation (OS) or in vitro fertilisation (IVF) improves pregnancy rates compared to expectant management. Expectant management in couples with unexplained infertility leads to live birth rates varying between 10 and 50 % per year, depending on their prognosis, based on female age, duration of subfertility and the outcomes of the fertility workup. Optimal implementation of expectant management in couples with good prospects of natural conception can prevent unnecessary treatments, complications and costs.
It is widely accepted that immunological infertility is one of the major causes of infertility in humans. On average, infertility occurs in one in every five couples of reproductive age and about 10-20 % of them is unexplained. A subset of infertile patients has been found to possess antisperm antibodies (ASA) in semen, blood, cervical mucus, or follicular fluid. ASA may affect sperm concentration and survival, limit sperm transport and motility and therefore the passage through the female reproductive tract specifically the cervix, inhibit sperm oocyte binding, prevent capacitation and acrosome reaction, affect embryo development and implantation, and has been proposed to lead to spontaneous miscarriage. Several strategies are used to counteract the deleterious effects of ASA-mediated infertility. The basic approaches for the treatment of men with ASA are as follows: minimizing exposure to sperm antigen like the use of condoms, decrease ASA production by immunosuppression with prednisone, removal of sperm bound ASA by a combination of laboratory techniques and sperm washing, and finally the most successful of all, the assisted reproductive technologies (ART).
Approximately 15 % of couples of reproductive age are affected by infertility. For couples, male factor contributes in up to half of these cases of infertility. Up to 25 % of infertile men will have no identifiable cause for their abnormal semen analyses. In this chapter, we discuss the potential indications, mechanism of action, and therapy options for empiric medical therapy of male unexplained infertility. Specific therapy regiments reviewed include selective estrogen receptor modulators such as clomiphene citrate, aromatase inhibitors, and injectable gonadotropins. We additionally discuss the negative impact of exogenous androgens on sperm production.
Ovulation induction with clomiphene citrate (CC) alone or combined with intrauterine insemination (IUI), when indicated, is an effective and relatively inexpensive first line treatment for "unexplained infertility" due to suboptimal ovulation or cervical mucus factor, and may shorten the time to pregnancy by increasing the number of preovulatory follicles. Its most effective use will be in women older than 30 years because of the age-related decrease in antral follicles and in women with suboptimal ovulation as evidenced by low preovulation estrogen levels and low progesterone levels, or a short luteal phase. Outcomes are dependent on preovulatory follicle numbers and age of the female partner. Treatment should be continued for a maximum of four cycles except in women with ovulatory dysfunction where it may be continued for up to six cycles. Pregnancy rates can be improved and the risk of multiple pregnancies reduced by ultrasound and hormone monitoring.
When considering treatment options for couples with unexplained infertility the initial approach often involves intrauterine insemination (IUI) combined with ovulation induction. Aromatase inhibition is a newer method of ovulation induction that has been used and studied as an alternative to clomiphene citrate (CC) to treat patients with unexplained infertility. Letrozole has been studied for its use as an adjunct to assisted reproductive technologies in an attempt to minimize the dose of costly gonadotropins needed and to overcome the adverse peripheral effects of clomiphene on the endometrium. Use of letrozole may eventually supersede that of clomiphene if the results of ongoing multicenter trials provide definitive evidence that letrozole has similar efficacy to gonadotropins or CC with a favorable side-effect profile and reduced cost.
The use of gonadotropins in the treatment of unexplained infertility for women, who are apparently ovulating regularly, appears logically to be a futile exercise. However, whether by correcting subtle deficiencies in the ovulation process, improving the endocrine environment, and/or endometrial receptivity, ovarian stimulation with gonadotropins produces better pregnancy rates than either clomiphene citrate alone or intrauterine insemination (IUI) alone. When gonadotropin stimulation was combined with IUI, it produced superior pregnancy rates than either clomiphene citrate and IUI or IUI alone. It has been suggested, however, that gonadotropin stimulation combined with IUI does not produce superior birth rates compared with expectant treatment. Due to a lack of evidence-based studies in this field, the use of gonadotropin stimulation in the treatment of unexplained infertility, although now widely employed, remains controversial and further well-designed studies are needed.
The introduction of gonadotropin therapy in medical practice represented an essential upgrade in infertility treatment. Follicle stimulation hormone (FSH) has a major role in follicular recruitment and growth. Improvements in purification techniques and recombinant technology have led to the two main FSH currently available: highly purified human menopausal gonadotropin (HP-hMG), with 1:1 ratio of FSH/luteinizing hormone (LH) activity; and recombinant human FSH (rec-hFSH), with only FSH activity. Both of them are safe to use and have overall similar clinical efficacy. However, rec-hFSH is purer than HP-HMG. Moreover, the introduction of filled-by-mass technology virtually eliminated batch-to-batch variation, enabled FSH accurate dosing and the development of novel prefilled pen devices that made treatment more patient-friendly. LH supplementation has also been introduced in controlled ovarian stimulation (COH), initally in hypogonadotropic hypogonadism women and then, in older patients (≥ 35 years), poor and slow/hypo responders and those with deeply suppressed endogenous LH. At present, three formulations with LH activity are available (i) hMG in which LH activity is given by hCG, (ii) recombinant human LH (rec-hLH), and (iii) fixed combination of rec-hFSH and rec-hLH at 2:1 ratio. Recent advances in the therapeutical options for COH include the introduction of the long acting FSH and the new family of pen injectors. Research now focuses on the development of small orally bioactive agonists of FSH and LH receptors that may in the future replace gonadotropin injections.
Unexplained infertility (UI) is diagnosed when the standard work-up reveals normal semen analysis, normal uterus with bilateral tubal patency, and regular ovulatory cycles. The true prevalence is still debatable but it is generally accepted to affect about 20 % of infertile couples. The treatment of UI is empirical since the true reasons of the reproductive dysfunction(s) are unknown. Expectant management (EM) with proper instructions to time intercourse could be suggested as first step in the treatment algorithm, followed by intrauterine inseminations (IUIs), with or without controlled ovarian stimulation (COS). After failed cycles of IUI, assisted reproductive technologies as conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) are the proposed subsequent steps. The gonadotrophin-stimulated IUI cycles are considered a better option than natural cycles IUI. A recent Cochrane review showed that the addition of COS to IUI treatment improves live-birth rates (LBRs) in couples with UI. Likewise, a small but still statistically significant increase in pregnancy rate was found with IUI when compared with timed intercourse (TI) in stimulated cycles. The overall success rate in couples with UI is high. However, the criteria for deciding when to suggest IUI or IVF need to be investigated in randomized controlled trials.
There is no established definition for "unexplained female infertility." Nevertheless, various treatment modalities have been offered to couples in whom no cause of infertility was found after thorough evaluation. Evidence shows that in vitro fertilization (IVF) is an effective treatment for unexplained infertility, and is associated with higher clinical pregnancy and live birth rates compared to expectant management. Current evidence also shows that intrauterine insemination (IUI) with or without controlled ovarian hyperstimulation (COH) may be equally effective in treatment-naïve patients, but this information is based on small studies and larger randomized controlled trials (RCTs) are needed. In pretreated patients, IVF is associated with significantly higher clinical pregnancy and live birth rates compared to IUI. Performing ICSI and IVF on sibling oocytes can help clarify the cause of infertility, and ICSI is associated with a significantly higher fertilization rate compared to standard IVF. The cost of achieving a live birth through IVF is higher than with IUI, but this should be seen in the light of the time needed to achieve the pregnancy.
The inability of healthcare provider to conclusively identify the cause of infertility in couples leads to the designation of unexplained infertility. It is likely that abnormalities leading to infertility exists in couples but not detected by the current methods and the state of technology. Normal semen parameters in men reduce but do not completely eliminate the probability of male infertility. The wide range in World Health Organization (WHO) reference values of semen parameters, repeated revisions of normal reference range over the years, significant variation in interlaboratory assessment of sperm characteristics, and use of different analytical criteria (WHO versus Krueger’s Strict morphology) further complicates the clinical assessment of normal semen parameters. High incidence of chromosomal aberrations and/or high percentage of DNA damage in sperm, oxidative stress, exposure of men to environmental pollutants/toxins, etc. may be likely factors that can be the suspected cause of unexplained infertility. Abnormal expression of certain sperm proteins may play a role in failure of fertilization resulting in unexplained male infertility at clinical level. Unexplained male infertility is strongly suspected in couples if the female partner presents with the history of successful pregnancy in not too distant past and subsequent comprehensive diagnostic tests fail to lead to a confirmed infertility diagnosis.
Unexplained male infertility (UMI) remains a challenge to both the physician and the patient. Currently, the management options for unexplained infertility, beyond assisted reproductive techniques, are typically limited to empiric therapies. This chapter focuses on stem cell biology and gene therapy that may offer potential treatment options for the most severe forms of male infertility, including idiopathic infertility, in the future. While stem cells and gene therapy have made some interesting advances in animal studies, there is still a need for additional research before these developments can be translated to the clinical setting.
... Many factors can influence sperm parameters, such as age, lifestyle, genetic and environmental exposure (Agarwal et al., 2020). The decline in semen quality is assumed to be directly related to environmental risk factors rather than pre-existing conditions (Maartens et al., 2015;Schattman et al., 2015). Increasing evidences suggest the associations between semen quality and the urine/blood concentrations of metals or metalloids (collectively referred to as "metals"). ...
Environmental metal exposure has been associated with decreasing semen quality, but the effects of multiple metal exposure on seminal plasma metabolome remain obscure. In this study, semen and repeated urine samples from 551 volunteers were collected in Wuhan City. Heavy metals and trace elements were measured using inductively coupled plasma mass spectrometer, and seminal plasma metabolomes were acquired using liquid chromatography coupled with high-resolution mass spectrometry. Weighted gene co-expression network analysis showed more than half of the seminal plasma metals were associated with specific metabolite modules, whereas only a few urine metals presented weak associations, indicating that seminal plasma may be an ideal biological sample for male reproductive biomarker discovery and exposure risk assessment. Seminal plasma zinc (Zn) and selenium (Se) concentrations were significantly associated with 22 metabolites (e.g., glycerophospholipids, acyl-carnitines and amino acid derivatives). Among these metabolites, acyl-carnitines were positively associated with semen quality and sperm concentration. Moreover, acyl-carnitines were associated with both Zn and Se exposure, indicating the potential role of carnitine pathway in their toxicity mechanism. Our findings suggest that seminal plasma metabolome connects Zn and Se exposure and sperm concentrations in Chinese men of reproductive age.
There has been emerging research linking per- and poly-fluoroalkyl substances (PFAS) to gamete viability and fertility. PFAS, prevalent in the environment and water supplies, undergo slow degradation due to their C-F bond and a long half-life (2.3–8.5 years). In females, PFAS inhibit the hypothalamic–pituitary–gonadal (HPG) axis, reducing follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, leading to the inhibition of androgen and estradiol production. PFAS have been found to cause detrimental effects on egg quality through impairing folliculogenesis. In males, PFAS can impair sperm motility and morphology: two fundamental qualities of successful fertilization. PFAS exposure has been proven to inhibit testosterone production, sperm capacitation, and acrosomal reaction. After fertilization, the results of PFAS exposure to embryos have also been investigated, showing reduced development to the blastocyst stage. The aim of this review is to report the main findings in the literature on the impact of PFAS exposure to gamete competency and fertilization capability by highlighting key studies on both male and female fertility. We report that there is significant evidence demonstrating the negative impacts on fertility after PFAS exposure. At high doses, these environmentally abundant and widespread compounds can significantly affect human fertility.
The global trend of rising (male) infertility is concerning, and the unidentifiable causes in half of the cases, the so-called unknown origin male infertility (UOMI), demands a better understanding and assessment of both external/internal factors and mechanisms potentially involved. In this work, it was our aim to obtain new insight on UOMI, specifically on idiopathic (ID) and Unexplained male infertility (UMI), relying on a detailed evaluation of the male gamete, including functional, metabolic and proteomic aspects. For this purpose, 1114 semen samples, from males in couples seeking infertility treatment, were collected at the Reproductive Medicine Unit from the Centro Hospitalar e Universitário de Coimbra (CHUC), from July 2018–July 2022. Based on the couples’ clinical data, seminal/hormonal analysis, and strict eligibility criteria, samples were categorized in 3 groups, control (CTRL), ID and UMI. Lifestyle factors and anxiety/depression symptoms were assessed via survey. Sperm samples were evaluated functionally, mitochondrially and using proteomics. The results of Assisted Reproduction Techniques were assessed whenever available. According to our results, ID patients presented the worst sperm functional profile, while UMI patients were similar to controls. The proteomic analysis revealed 145 differentially expressed proteins, 8 of which were specifically altered in ID and UMI samples. Acrosin (ACRO) and sperm acrosome membrane-associated protein 4 (SACA4) were downregulated in ID patients while laminin subunit beta-2 (LAMB2), mannose 6-phosphate isomerase (MPI), ATP-dependent 6-phosphofructokinase liver type (PFKAL), STAR domain-containing protein 10 (STA10), serotransferrin (TRFE) and exportin-2 (XPO2) were downregulated in UMI patients. Using random forest analysis, SACA4 and LAMB2 were identified as the sperm proteins with a higher chance of distinguishing ID and UMI patients, and their function and expression variation were in accordance with the functional results. No alterations were observed in terms of lifestyle and psychological factors among the 3 groups. These findings obtained in an experimental setting based on 3 well-defined groups of subjects, might help to validate new biomarkers for unknown origin male infertility (ID and UMI) that, in the future, can be used to improve diagnostics and treatments.
This study aims to determine the association of non-essential trace elements present in follicular fluid, plasma, and urine with reproductive outcomes of women undergoing intracytoplasmic sperm injection (ICSI), preimplantation genetic testing for aneuploidies (PGT-A) and single frozen euploid embryo transfer (SET/FET). This single-center, prospective cohort study included sixty women undergoing ICSI with PGT-A and SET/FET between 2018 and 2019. Urine, plasma and follicular fluid samples were collected on the vaginal oocyte retrieval day to simultaneously quantify ten non-essential trace elements (i.e., Ba, Sr, Rb, Sn, Ti, Pb, Cd, Hg, Sb, and As). We found several associations between the levels of these non-essential trace elements and clinical IVF parameters. Specifically, the increased levels of barium in follicular fluid were negatively associated with ovarian function, pre-implantation development and embryo euploidy, while elevated strontium concentrations in this biofluid were negatively associated with impaired blastulation and embryo euploidy. Elevated plasma strontium levels were negatively associated with ovarian function, fertilization and blastulation. Enhanced presence of other trace elements in plasma (i.e., rubidium and arsenic) were associated with a diminished ovarian function and limited the number of recovered oocytes, mature oocytes and zygotes, respectively. Fully adjusted models suggested significantly lower odds of achieving a live birth when increased concentrations of barium and tin were found in urine.
Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells’ viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.
BACKGROUND: The excessive concentration of leptin has negative effects on all aspects of female reproduction. Despite this established relationship, the exact role of leptin in women’s fertility is not clear enough and needs more clarification.
AIM: To evaluate the serum leptin levels in Sudanese women and to ascertain the relationship between serum leptin levels and unexplained infertility (UI).
METHODS: A matched (age and body mass index) case-control study was conducted from March 2021 to February 2022. The study samples were 210 women with UI and 190 fertile women of reproductive age who were attending the maternity hospitals and fertility clinics in Khartoum state Sudan. The serum concentration of leptin and other serum biomarkers were determined using enzyme-linked immunosorbent assays.
RESULTS: The results showed that there was a highly statistically significant difference between the two groups (P < 0.001) for all examined eight biomarkers. Whereby, leptin, luteinizing hormone (LH)/follicular stimulating hormone (FSH) ratio, prolactin hormone (PRL), and testosterone (T) were significantly higher in the UI group compared with the control group. In contrast, FSH and estradiol (E2)/T ratio were significantly lower in the UI group than in the control group and the effect size test for the difference between the two groups was very large (effect size > 0.80), for leptin level, LH/FSH ratio, PRL level, and E2/T ratio, and large (effect size 0.50- ≤ 0.80) for FSH and T.
CONCLUSION: This study reveals that leptin could be a potential biomarker for UI in Sudanese women and it may be useful for identifying women with a high risk of infertility.
Antioxidant supplementation in idiopathic male infertility has a beneficial effect on semen parameters. However, the molecular mechanism behind this effect has not been reported. The objective of this study was to evaluate the sperm proteome of idiopathic infertile men pre- and post-antioxidant supplementation. Idiopathic infertile men were provided with oral antioxidant supplementation once daily for a period of 6 months. Of the 379 differentially expressed proteins (DEPs) between pre- and post-antioxidant treatment patients, the majority of the proteins (n = 274) were overexpressed following antioxidant treatment. Bioinformatic analysis revealed the activation of oxidative phosphorylation pathway and upregulation of key proteins involved in spermatogenesis, sperm maturation, binding of sperm, fertilization and normal reproductive function. In addition, the transcriptional factors associated with antioxidant defense system (PPARGC1A) and free radical scavenging (NFE2L2) were predicted to be functionally activated post-treatment. Key DEPs, namely, NDUFS1, CCT3, PRKARA1 and SPA17 validated by Western blot showed significant overexpression post-treatment. Our novel proteomic findings suggest that antioxidant supplementation in idiopathic infertile men improves sperm function at the molecular level by modulating proteins involved in CREM signaling, mitochondrial function and protein oxidation. Further, activation of TRiC complex helped in nuclear compaction, maintenance of telomere length, flagella function, and expression of zona pellucida receptors for sperm–oocyte interaction.
Up to 30% of men with normal semen parameters suffer from infertility and the reason for this is unknown. Altered expression of sperm proteins may be a major cause of infertility in these men. Proteomic profiling was performed on pooled semen samples from eight normozoospermic fertile men and nine normozoospermic infertile men using LC-MS/MS. Furthermore, key differentially expressed proteins (DEPs) related to the fertilization process were selected for validation using Western blotting. A total of 1139 and 1095 proteins were identified in normozoospermic fertile and infertile men, respectively. Of these, 162 proteins were identified as DEPs. The canonical pathway related to free radical scavenging was enriched with upregulated DEPs in normozoospermic infertile men. The proteins associated with reproductive system development and function, and the ubiquitination pathway were underexpressed in normozoospermic infertile men. Western blot analysis revealed the overexpression of annexin A2 (ANXA2) (2.03 fold change; P = 0.0243), and underexpression of sperm surface protein Sp17 (SPA17) (0.37 fold change; P = 0.0205) and serine protease inhibitor (SERPINA5) (0.32 fold change; P = 0.0073) in men with unexplained male infertility (UMI). The global proteomic profile of normozoospermic infertile men is different from that of normozoospermic fertile men. Our data suggests that SPA17, ANXA2, and SERPINA5 may potentially serve as non-invasive protein biomarkers associated with the fertilization process of the spermatozoa in UMI.
Objective: To test the effect of voltage-gated sodium channels (VGSCs) blockers on the
motility and viability of human sperm in-vitro and to evaluate the tested compounds as
potential contact spermicidal. Methods: Sperm samples were obtained from healthy nonsmoking
volunteers of age 25-30 years who had not taken any drug 3 months before
and during the course of the study. The effect of VGSCs blockers evaluated from two
pharmacological classes including antiarrhythmic (amiodarone, procainamide and
disopyramide) and antiepileptic (carbamazepine, oxcarbazepine, phenytoin, and lamotrigine)
drugs. They were tested on the in-vitro motility and viability of human sperm using Computer
Assisted Semen Analyzer. Results: All tested drugs except oxcarbazepine showed dose
dependent inhibition of total motility with significant reduction (P
Prolonged use of immunosuppressant medications is occasionally seen in infertile men with chronic inflammatory conditions; autoimmune disorders; or an organ or hematopoietic stem cell transplant. Chronic inflammation impacts negatively on male reproductive endpoints, so immunosuppressant therapy can produce improvements. Corticosteroids have been used to treat antisperm antibodies and even as an empirical treatment for male infertility in general. Trials of these methods have provided mixed results on semen quality and fertility, with improvement, no change and negative effects reported by different investigators. In a substantial number of observational studies, patients on long-term therapy with prednisone for chronic inflammatory disease, testosterone levels were lower compared to untreated controls, though randomized controlled trials have not been conducted. Similarly decreases in testosterone have been reported in men receiving corticosteroids to minimize transplant rejection; however, most were treated with multiple immunosuppressive medications that may have contributed to this effect. A large number of trials of healthy men treated with corticosteroids have shown some disruption in reproductive hormone levels, but other studies reported no effect. Studies in monkeys, rats (at human equivalent dose), cattle, sheep, and horses have shown endocrine disruption, including low testosterone with dexamethasone treatment. Of the cytostatic immunosuppressives, which have high potential for cellular damage, cyclophosphamide has received the most attention, sometimes lowering sperm counts significantly. Methotrexate may decrease sperm numbers in humans and has significant negative impacts in rodents. Other chemotherapeutic drugs used as immunosuppressants are likely to impact negatively on male fertility endpoints, but few data have been collected. The TNF-α Inhibitors have also received little experimental attention. There is some evidence that the immunophilin modulators: cyclosporine, sirolimus, and everolimus cause endocrine disruption and semen quality impairment. As we review in this chapter, results in experimental species are concerning, and well-designed studies are lacking for the effects of these medications on reproductive endpoints in men.
Study question:
Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF), and unexplained (UE)?
Summary answer:
The global blastocyst transcriptome was significantly altered in association with a PCO, MF, and UE infertility diagnosis.
What is known already:
Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during pre-implantation development.
Study design, size, duration:
Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF, and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control).
Participants/materials, setting, methods:
Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were < 38 years old with male patients < 40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50), and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR).
Main results and the role of chance:
Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348, and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion, and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9), and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold).
Large scale data:
Not available due to participant privacy.
Limitations, reasons for caution:
Blastocyst samples for microarray analysis required pooling. While this allows for an overall average in each infertility etiology group and can reduce noise from sample-to-sample variation, it cannot give a detailed analysis of each blastocyst within the group.
Wider implications of the findings:
Underlying patient infertility diagnosis has an impact on the blastocyst transcriptome, modifying gene expression associated with developmental competence and implantation potential.
Study funding and competing interest(s):
No conflict of interest or outside funding provided.
Thirteen mammalian aquaporin (AQP) water channels are known, and few of them play a role in the mammalian reproductive system. In our earlier study, the predominance of AQP5 in the late-pregnant rat uterus was proven. Our current aim was to investigate the effect of estrogen- and gestagen-related compounds on the expression of the AQP5 channel in the late-pregnant rat uterus. Furthermore, we examined the effect of hormonally-induced preterm delivery on the expression of AQP5 in the uterus. We treated pregnant Sprague-Dawley rats subcutaneously with 17β-estradiol, clomiphene citrate, tamoxifen citrate, progesterone, levonorgestrel, and medroxyprogesterone acetate. Preterm delivery was induced by subcutaneous mifepristone and intravaginal prostaglandin E2. Reverse-transcriptase PCR and Western blot techniques were used for the detection of the changes in AQP5 mRNA and protein expressions. The amount of AQP5 significantly increased after progesterone and progesterone analogs treatment on 18 and 22 days of pregnancy. The 17β-estradiol and estrogen receptor agonists did not influence the AQP5 mRNA level; however, estradiol induced a significant increase in the AQP5 protein level on the investigated days of gestation. Tamoxifen increased the AQP5 protein expression on day 18, while clomiphene citrate was ineffective. The hormonally-induced preterm birth significantly decreased the AQP5 level similarly to the day of delivery. We proved that AQP5 expression is influenced by both estrogen and progesterone in the late-pregnant rat uterus. The influence of progesterone on AQP5 expression is more predominant as compared with estrogen.
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