“Causes of Male Infertility: A 9-year Prospective Monocentre Study on 1737 Patients with Reduced Total Sperm Counts” (2016), by Margus Punab, Oliv Poolamets, Piia Paju, Vladimir Vihljajev, Kristjan Pomm, Ruth Ladva, Paul Korrovits, and Maris Laan

In December of 2016, Margus Punab and colleagues published “Causes of Male Infertility: A 9-year Prospective Monocentre Study on 1737 Patients with Reduced Total Sperm Counts,” hereafter “Male Infertility,” in the journal Human Reproduction. The study examines the main causes of severe male factor infertility, or SMF infertility, which occurs when a male’s semen has a very low number of healthy sperm cells or contains atypically low levels of sperm cells. In “Male Infertility,” the authors determine the primary cause of SMF infertility in forty percent of their participants, and among those participants, they found that the primary causes of SMF infertility were varicoceles, or enlarged veins within the loose bag of skin holding the testicles. The authors did not determine the cause of SMF infertility in the remaining sixty percent of the cases, noting a gap in the current understanding of the causes of SMF infertility. “Male Infertility” was one of the first large-scale studies to reveal certain underlying causes of SMF infertility, and its conclusions have allowed researchers to investigate fertility solutions for male patients who would otherwise not be able to reproduce.

1. Background and Context
2. Article Roadmap
3. Detailed Contents
4. Impact

Background and Context

At the time of publication, each of the authors of “Male Infertility” worked at the University of Tartu and the Tartu University Hospital, located in Tartu, Estonia, which, according to its own webpage, as of 2024, is the largest healthcare provider in Estonia. Seven of the authors worked in the Andrology Center at the hospital, and andrology is the branch of medicine that deals with the male reproductive system, including male infertility issues. The senior author, Maris Laan, has affiliations with the Institute of Molecular and Cell Biology and the Institute of Biomedicine and Translational Medicine at the University of Tartu. At the time of the article’s publication, the primary author, Punab, studied male reproductive function, infertility, and diseases of the prostate, which is the male organ that produces seminal fluid. The senior author, Laan, specializes in reproductive medicine and genetics and works as a professor of human genetics at the Institute of Biomedicine and Translational Medicine, as of 2024.

In “Male Infertility" the authors discuss various terms related to fertilization and their relation to male infertility. During fertilization, a sperm cell joins with an egg, the female reproductive cell. Semen, or seminal fluid, which is the fluid that transports the sperm to the female egg, facilitates that process. Once one sperm cell fertilizes the egg cell, it forms a zygote. The zygote then travels through the fallopian tube to the uterus where it will become an embryo and eventually a fetus, at eight weeks after fertilization, for the duration of pregnancy. Infertility in either the male or female partner causes the couple to be unable to have a child through sexual intercourse alone.

The authors also discuss possible defects in sperm that could lead to male infertility. One such characteristic that physicians commonly see associated with male infertility is a low sperm count. A low number of sperm cells makes it less likely that a sperm can reach and enter an egg. Additionally, physicians commonly see abnormal morphology, or misshaped sperm, in patients experiencing male infertility. To enter the outer layer of the egg cell, a sperm must be the proper shape. Lastly, a lack of sperm motility, or the ability of sperm to move efficiently, is another very common characteristic of male infertility. Ineffective sperm motility makes it difficult for the sperm to make the journey through the female reproductive system to reach the egg. Still, many other issues can cause a male to be infertile such as a blockage preventing the sperm from releasing and the sperm having trouble attaching to the egg.

When researchers first discovered SMF infertility, no cure existed, meaning males with SMF infertility simply could not have children, but since that time, researchers have developed treatments for fertility. In 1978, the first baby was born via in vitro fertilization, or IVF. IVF is a form of assisted reproductive technology, or ART, which encompasses all treatments that aid couples who are experiencing infertility, including male infertility. Another type of ART is intracytoplasmic sperm injection, or ICSI. ICSI is a part of IVF where the doctors harvest sperm cells from the male and egg cells from the female, then combine them in a laboratory and implant the fertilized egg back into the female.

Article Roadmap

The article “Male Infertility” consists of five main sections, some with subsections. In the “Introduction,” the authors explain that fifteen percent of couples reportedly are unable to conceive after a year, and fifty percent of those couples have difficulty conceiving because of male infertility. In the second section, “Materials and Methods,” the researchers explain that they studied 1,737 males with varying levels of SMF infertility and 325 males with typical fertility to determine the differences in the sperm between the two groups of males. The authors discuss that they also performed genetic tests and hormone assays to determine the causes of SMF infertility. In “Results,’’ they write that they could only diagnose the primary causes of infertility in forty percent of the males they studied and compared the primary causes and potential contributing factors for all groups. They also discuss that the other sixty percent had idiopathic, or unknown, causes, and the authors established that they had less success with figuring out the causes of the SMF infertility in males with more severe cases. In “Discussion,” the authors explain that their study was comprehensive because they studied more than ninety percent of all cases of SMF infertility in Estonia, but they propose that researchers should continue to conduct further research on the causes of SMF infertility in the participants their workup could not diagnose. In “Conclusions and Perspectives,” the authors reiterate that uncovering the causes and mechanisms behind SMF infertility can lead to better management and potential solutions to SMF infertility in males.

Detailed Contents

In “Introduction,” Punab and colleagues discuss that previous studies inadequately assessed male infertility and that, therefore, their study focused on understanding the causes of male infertility through semen analysis. The authors state that about fifteen percent of couples actively trying to conceive are not able to within a year. They note that the problem for about fifty percent of those couples is male infertility related to semen, such as poor semen quality. The authors explain that in their study, they compared the sperm of 1,737 individuals with SMF infertility to the sperm of 325 fertile individuals to determine whether the known causes of general male infertility are also the causes of SMF infertility. They write that they organized the SMF infertility patients into groups according to the severity of their SMF infertility to determine the primary causes of SMF infertility within each group. They claim that revealing the causal factors of SMF infertility will allow researchers and physicians to better understand it and work towards new methods of treatment.

Also in the “Introduction,” Punjab and colleagues note that they chose to analyze the underlying causes of male infertility because ART treats patient’s infertility symptoms, rather than the causes. They explain that they did that because the increase in popularity and refining of ART has led to a decrease in searching for the underlying causes of the male infertility. According to the authors, they addressed the underlying issues that result in male infertility so that other researchers could create treatments for those root causes. The authors state that prior studies lacked either large-scale findings or comparisons of data to that of a control group. Thus, they conducted “Male Infertility” as a long-term study based at a single location.

In the “Materials and Methods,” Punab and his colleagues discuss the demographics and requirements for the experimental and control groups of their study. The authors explain that they recruited the participants of the experimental and control groups between January 2005 and December 2013. They state that they investigated a total of 8,518 males with infertility issues over that time frame, but only 1,737 of those males had SME infertility and participated in the study. The authors also discuss that they studied a control group of 325 males who had a pregnant partner so that they could compare the differences in sperm between members of the infertile group and fertile group. The authors clarify that they classified participants with SMF infertility as individuals who had severely low amounts of sperm, less than 39 million cells, in their semen for at least two semen analyses in a row. Additionally, the researchers explain that they only classified a participant as having SMF infertility if they and their partner had tried and failed to conceive for over twelve months.

The authors explain that they performed semen analysis, physical examinations, medical interviews, and blood tests to fully analyze and compare both the experimental and control groups. The research team also performed genetic tests for 1,216 individuals of the infertile group who had sperm concentrations less than 5 million per milliliter. The researchers explain that when analyzing the sperm samples from each patient, they measured the semen volume, sperm concentration, sperm shape, and sperm motility, or the ability of the sperm to move efficiently. In addition, they write that they used semen smears to analyze the morphology, or shape, of the sperm cells, and the amount of white blood cells present because white blood cells can be a sign of infection or inflammation, which can contribute to infertility.

Next, the research group discusses that they separated their 1,737 participants in the SMF infertility group into five groups based on the severity of their sperm impairment. They write that the most severe group had aspermia, which means the participants did not produce sperm or semen upon ejaculation, and the second most severe group had azoospermia, which means that the participants produced semen when they ejaculated, but the semen did not contain any sperm. The authors explain that the other three groups contained participants who produced semen containing various levels of sperm when they ejaculated. They state that those with cryptozoospermia had a sperm count of less than 1 million per ejaculation, those with severe oligozoospermia had between 1 million to 10 million sperm per ejaculation, and those with severe oligospermia had a sperm count between 10 million to 38 million. In their article, the authors include a table showing that just over 2.5 percent of the participants had aspermia, twenty-two percent had azoospermia, approximately seven and a half percent had cryptozoospermia, almost twenty-one percent had severe oligozoospermia, and almost forty-seven percent had moderate oligozoospermia as their severity of SMF infertility.

In a subsection of “Materials and Methods” titled “Clinical Examination,” the authors explain that when clinicians conducted physical examinations for each patient, they looked for infertility indicators such as genital anatomy and testicular size. They write that the clinicians measured both the right and left testicles and found the sum to determine the total volume of the testes. The authors describe that for each participant, the clinicians also documented the position of the testicles in relation to the scrotum, the pathology of the genital ducts, and the pathology of the penis. The scrotum is a sac of skin located below the penis that contains the testes and other parts of the male reproductive system. Then, the researchers explain that the clinicians noted if each patient had any varicoceles, which are enlarged veins in the scrotum that can be indicative of problems related to infertility such as low sperm production or testicle developmental issues. They write that if the clinicians noted a varicocele, they graded it on a scale of one to three, with grade three being the most serious. Finally, the authors discuss that during the examinations, the clinicians investigated whether each participant had cryptorchidism, a condition where one or both testes do not lower into the scrotum from the abdomen. They note that those examinations were important for them to identify which participants had physical signs correlated with infertility issues and to find any other similarities between participants in the same SME infertility group.

Next, Punab and colleagues discuss that clinicians also performed additional tests, such as hormone and genetic analyses, to determine the causes of SMF fertility in the participants and investigate potential correlations across the five subgroups of SMF infertility. First, they write that they drew blood to examine the levels of the male sex hormones, including FSH, LH, and testosterone. Those hormones are important for fertility, so obtaining a patient’s levels assisted the researchers in identifying the cause of the SMF fertility. The authors explain that clinicians also conducted a genetic test known as karyotype analysis for each participant. Karyotyping is a type of test that helps to identify genetic problems or abnormalities within an individual that may be causing a disease or disorder. In addition, the authors state that they screened the participants for any Y-chromosome microdeletions. When Y-chromosome microdeletions occur, it affects sperm production and commonly causes male infertility.

Continuing in “Materials and Methods,” the authors distributed the causal factors of SME infertility into six groups, including genetic and clinical factors. The authors state that they assigned each of the patients in the study to one primary cause of infertility that they then ranked from most to least influential. The researchers discuss that they could only link forty percent of SMF infertility participants to the six primary casual factors, so they classified the other sixty percent of cases as idiopathic, or unknown infertility. They found that the most influential causal factor for SME infertility was genetic causes, which accounted for almost eight percent of participants. They explain that 1.3 percent of the participants had secondary hypogonadism, which is when the testicles appear to be normal but do not function properly due to a problem in the parts of the brain that release hormones important for fertility, as their primary causal factor. The researchers describe congenital anomalies to be the third most influential causal factor for SME infertility, and that accounted for 10.7 percent of participants in the study. They classified the fourth most influential causal factor as oncological diseases, or cancer, which included patients who had received systematic chemotherapy, radiotherapy, or an operative treatment for their cancer that negatively affected their genital tract in a way that caused SMF infertility. They write that 3.4 percent of participants had oncological diseases as their primary causal factor. The authors identified that severe sexual dysfunctions accounted for 4.4 percent of the participants’ primary causal factors of SMF infertility. Lastly, they explain that the primary causal factor for almost thirteen percent of participants was testicular factors, such as trauma to the testis and operations on the genital tract, that resulted in decreased testicular volume and ultimately SMF infertility. The authors note that twenty-eight of the SMF infertility cases with known causes had more than one causal factor, and in those cases, they assigned the case to the causal factor that was more influential.

The authors also acknowledge clinical factors that they claimed only potentially contribute to SMF infertility, such as surgical history and physiology. The researchers call those factors potential contributors because although researchers know that they sometimes have harmful effects on fertility, they often do not lead to infertility. For example, the authors explain that varicoceles, testicular traumas, hernia operations, unusually high amounts of white blood cells in the semen, chronic disease, and obesity can sometimes but do not always cause infertility when present. The authors define chronic diseases as non-genital diseases that have lasted for more than three months. Some examples of those include asthma, depression, cardiovascular diseases, and neurological disorders.

In “Results,” the authors state that their findings suggest that there were differences in sperm count and hormone levels for patients with SMF infertility compared to the control group. They explain that they found that, on average, patients with SMF infertility had lower sperm concentration and sperm counts than patients in the control group. They write that the average sperm concentration in the SMF infertility group was nearly twenty-five times lower than the average sperm concentration of the control group. Additionally, they reveal that the total sperm count for the infertility group was thirty-two times lower than the total sperm count in the semen of the control group. The researchers also state that they found significantly higher levels of LH hormones, on average, in the blood samples of SME infertility group compared to the control group. A high LH level can reveal that an individual’s sex organs do not produce enough of the hormones needed for certain reproductive processes to occur. Furthermore, the authors write that there was a significant difference between the FSH of the two groups as the SMF infertility group had, on average, almost three times higher levels of the hormone in their blood than the control group. An abnormally high FSH level can indicate a testicular problem with sperm production as it means the testicles are receiving an unusually high signal that sperm needs to be made.

Continuing in “Results,” Punab and colleagues note that, of the SMF infertility patients, about eighty-three percent had primary infertility while about seventeen percent had secondary infertility. Primary infertility in males occurs when the male has not successfully impregnated a female after a year of unprotected sex, while secondary infertility arises when a man has successfully fertilized an egg in at least one prior pregnancy, showing that they are not completely infertile, but is unable to at a later time. The authors explain that they found that eleven of the men in the control group with pregnant partners had generally accepted and known causal factors of male infertility, including testicular abnormalities or cancer, inflammation of the testicles, or mumps infection. From those results, the authors conclude that although those diagnoses are usually causal factors of infertility, they do not always cause infertility, especially when the causal factor only applies to one testicle.

Then, the authors also address that they found the primary causes of SMF infertility in only forty percent of the participants while sixty percent had idiopathic, or undetermined infertility, and they explain that there was a direct relationship between the severity of a patient’s case of SMF infertility and their ability to determine the patient’s causal factors. For example, the researchers explain that they determined the causal factors for all of the aspermia cases, the most severe group, but for moderate oligozoospermia, the least severe group, they were only able to identify the primary causal for twenty-two percent of patients, leaving about seventy-seven percent of the moderate oligozoospermia cases unidentified. The authors also discuss that within the five groups of SME infertility, the primary causes differed. For example, they write that in the aspermia group, severe sexual dysfunction explained nearly seventy-two percent of the cases, but for the azoospermia group, male secondary hypogonadism and seminal tract obstruction caused patient infertility in over eighty-five percent of all cases.

Finally, the authors explain that potentially contributing factors of chronic disease, overweight, and obesity impacted their ability to determine a participant’s primary causal factor while others, such as varicocele and leukocytospermia, did not. Leukocytospermia is the abnormal finding of leukocytes in human ejaculation. The researchers state that the proportion of patients with chronic disease, overweight, and obesity increased for the causal factor and idiopathic groups in comparison to the control group. They discuss that the idiopathic group did have a higher prevalence of grade two to three varicoceles and elevated white blood cell count in semen compared to the causal factor and control groups. The authors include a table that includes all the potential contributing factors that the authors explored and their prevalence among individuals in each of the five SMF infertility groups and individuals with each primary causal factor. That table shows that varicoceles and high white blood cell counts in the semen occur more in the participants with the causal factors of seminal tract obstruction and other testicular disorders. The authors suggest that their findings may reflect that varicoceles and high white blood cell counts could cause the seminal tract obstruction and testicular disorders that they noted as the primary causal factors.

In “Discussion,” the research group highlights the strengths of their study, including their sample size, study length, and methodology. They state that they were the first research team to conduct a large-scale, long-term, monocenter approach for studying SMF infertility. The authors report that their study evaluated over ninety percent of all SMF infertility cases in the country of Estonia. They write that they collected data over an extended period of time, nine years, and they included a control group to compare the results of individuals with SMF infertility to. In addition, the authors highlight that there is a large gap in researchers’ current understanding of the causes of SMF infertility. Thus, they advise future researchers to continue working on understanding the idiopathic SMF infertility cases.

In the fifth and final section, “Conclusions and Perspectives,” Punab and his colleagues assert that their study helped fill in large knowledge gaps about the causes of male infertility and acknowledge their limitations. The researchers report that their study investigated the role of the aspermia group in male infertility, which researchers previously knew very little about. However, the authors note that one limitation is that their study failed to identify the primary causal factor for sixty percent of SME infertility patients. They also acknowledge that their study only included males with reduced sperm counts, and their findings therefore are not generalizable to all male infertility cases. They also state that there is an urgent need for further research on infertility and SMF infertility to determine the causes and mechanisms behind oligozoospermia as it accounts for the largest number of idiopathic infertility cases. The authors suggest that uncovering that information could be important because it could guide improvements in the clinical management of the condition and potential solutions.

Impact

As of 2024, according to Google Scholar, researchers have cited “Male Infertility” over 460 times. The majority of those studies that cited “Male Infertility” attempted to build on the article to explain different aspects of male infertility. For example, in 2018, researchers at the Institute of Human Genetics at the University of Münster in Münster, Germany, published a study on the disorders of spermatogenesis, the process that produces sperm cells. Those researchers discovered three specific genes linked with male infertility and found that mutations within those genes are probably a cause of azoospermia in some males. Additionally, in 2018, researchers at the Department of Biomedical Sciences at the University of Antwerp in Antwerp, Belgium, published a study that focused on finding molecular mechanisms underlying male idiopathic infertility. The study did not find conclusive answers, and its authors report that male infertility is a complex issue with underlying factors that remain largely unknown. Those authors do, however, recommend that researchers further analyze both the molecular and epigenetic defects in spermatogenesis and sperm function to improve the individualized treatment options for men with idiopathic infertility.

The research team of “Male Infertility” aimed to help uncover the root causes of SMF infertility and encouraged continual research on the causes of idiopathic infertility rather than just treatments for it. The study reemphasized the prevalence of idiopathic infertility and found varicocele as the most common cause of SMF infertile men. “Male Infertility” dissected a large number of SMF infertility cases to give future researchers an idea of where large gaps exist in the academic understanding of the primary causes of infertility. As the authors state in “Male Infertility,” uncovering the causes of all cases of SMF infertility will allow researchers to work on ways to treat those problems rather than just focusing on assisted reproductive technologies which are expensive, invasive, and stressful for patients. According to a study in 2020, male infertility is a factor in about fifty percent of ART cases, and of those cases about twenty percent of the males are diagnosed with SMF infertility. Thus, the work of the authors of “Male Infertility” can contribute to future researchers investigating potential treatments of SMF infertility.