Male Infertility Evaluation and Sperm DNA Fragmentation

Male factors alone cause infertility in 30 to 35% of couples and contribute to almost half of all cases, yet a normal semen analysis does not prove that a man is fertile. A complete male infertility evaluation runs from a correctly collected semen analysis to hormonal, urological and genetic tests, and, in selected men, a sperm DNA fragmentation test, which measures damage to the genetic material inside the sperm.

Dr. Soma Singh, Associate Director - IVF & Infertility, Max Super Speciality Hospital, Noida, India, stresses that infertility is a combined problem in which male factors are equally responsible. This article sets out her approach to evaluating and treating the male partner. It belongs to a series that starts with recent advances in the management of an infertile couple; the woman's side of the evaluation is in the infertility workup.

Semen analysis: getting the sample right

Semen analysis is the first and simplest test for the man, and its value depends on how the sample is collected. Dr. Singh stresses that whoever orders the test must give the man clear instructions:

• The sample is given after 2 to 6 days of sexual abstinence.
• It is examined within one hour of ejaculation, ideally within minutes.
• It should therefore be collected close to the laboratory that will analyse it.

According to the WHO, the ideal method of semen analysis is manual examination under the microscope rather than automated or computer-assisted analysis.

WHO semen analysis reference values

Dr. Singh presented the lower reference limits from the current WHO manual. A result at or above each limit is considered normal:

Morphology describes the share of sperm with a normal shape, judged by the strict Tygerberg (Kruger) criteria; 4% normal forms or above is a normal result.

Why an abnormal result is repeated after about three months

An abnormal semen analysis is not a final verdict. Dr. Singh's rule of thumb is to repeat it after about 8 to 12 weeks, because sperm production takes almost 74 days and transport through the ducts another 15 or so. Any treatment, such as stopping smoking or a course of antioxidants, therefore shows its effect only around 90 days later.

Male infertility evaluation beyond semen analysis

A semen analysis shows whether the semen is abnormal, not why. The second-level evaluation has four arms: endocrine, urological, genetic and sperm function tests.

Endocrine evaluation

Hormone tests are indicated when the semen contains no sperm (azoospermia) or the concentration is below 10 million per mL, and also when the man has impaired libido, erectile dysfunction or small, atrophic testes. The first tests are:

• FSH and total testosterone. Testosterone peaks in the morning, so Dr. Singh asks for the sample between 7.30 and 8.30 a.m.

If total testosterone is low, below 300 ng/dL, the panel is extended:

• LH, free testosterone and a repeat FSH, to locate the problem in the pituitary or the testis.
• Prolactin, especially if he has headaches or gynaecomastia (breast enlargement).
• Oestradiol, if he is obese, particularly with central obesity, because a disturbed ratio of testosterone to oestradiol can itself cause abnormal semen.

Urological evaluation and imaging

A urologist or general surgeon who examines the man properly learns a great deal without imaging. When the examining gynaecologist is not confident of the male examination, Dr. Singh advises a scrotal ultrasound, which shows any scrotal mass, the position and volume of the testes and any varicocele (enlarged veins around the testis).

Transrectal ultrasound (TRUS) is reserved for suspected obstruction of the lower genital tract. The pattern that raises this suspicion is a low-volume ejaculate with no sperm or a very low count, in a man whose testes are of normal size and whose vas deferens can be felt. Vasography, an X-ray of the vas, is used only when surgery to bypass a block, such as vasovasostomy or vasoepididymostomy, is planned.

Obstructive or non-obstructive azoospermia?

The physical examination and blood tests distinguish the two forms of azoospermia. A man with normal-sized testes, a palpable vas and epididymis, normal hormones and no sperm almost certainly has an obstruction: sperm are made but cannot get out. A man with small testes and raised FSH has a production problem, called non-obstructive azoospermia, in which the testes are failing.

Genetic evaluation

Genetic tests are indicated in azoospermia and severe oligospermia (a very low count). The choice of test follows the type:

• Karyotype, an analysis of the number and structure of the chromosomes, is the most common test.
• Y-chromosome microdeletion testing in non-obstructive azoospermia and severe oligospermia. Deletions in the azoospermia factor (AZF) regions of the Y chromosome explain why a man produces few or no sperm and help predict whether sperm can be retrieved surgically.
• CFTR gene mutation testing when the vas deferens is absent, especially on both sides (congenital bilateral absence of the vas deferens). This condition is linked to mutations in the cystic fibrosis gene, and the female partner's carrier status then matters for the child.

Sperm function tests and reactive oxygen species

Many men remain infertile despite a normal semen analysis. Sperm function tests provide the clinically useful information that a routine analysis cannot. Of the available tests, Dr. Singh concentrates on two that are commercially available and that change management: sperm DNA integrity, and seminal reactive oxygen species. Others, such as the acrosome reaction and hyperactivated motility, remain research tools.

What oxidative stress does to sperm

Reactive oxygen species (ROS) are free radicals such as peroxide and hydroxyl radicals, produced continuously during the body's normal use of oxygen. An antioxidant system protects the body against the damage they cause. When ROS production outstrips antioxidant capacity, the resulting oxidative stress damages sperm function and can cause infertility.

Factors that increase ROS formation include:

• infection of the male accessory glands;
• smoking, drugs, pollution and radiation;
• varicocele;
• systemic disease such as diabetes, cancer or any chronic inflammatory disorder.

Sperm DNA fragmentation index (DFI) and what it means

ROS can be measured directly, but direct methods are mainly used in research. The practical, indirect measure is the DNA fragmentation index (DFI), which shows whether the DNA inside the sperm is intact. Sperm DNA is packed into the sperm head, a large nucleus with very little cytoplasm, bound to nuclear proteins, and its defences against free radical attack are weak. DNA integrity testing is therefore recommended as an additional, independent marker of male fertility.

Dr. Singh interprets the DFI in three bands:

Who should have a DFI test

Because the test is not available at every centre, Dr. Singh orders it selectively, for men with:

• advanced age;
• obesity or diabetes;
• smoking;
• varicocele;
• previous chemotherapy or radiotherapy;
• unexplained infertility, including the situation in which the woman has had children with a previous partner and cannot conceive with a new partner whose semen analysis is normal;
• recurrent pregnancy loss with his partner.

A case: unexplained infertility explained

A couple in their early thirties had tried to conceive for four years. All the woman's tests were normal, including her ovarian reserve. The man was a smoker, but his semen analysis was entirely normal. They had already had three cycles of intrauterine insemination (IUI) with ovulation-inducing drugs elsewhere, without success, and arrived with a working diagnosis of unexplained infertility.

His smoking was the one lead. Dr. Singh tested sperm DNA fragmentation, and his DFI was 42%. With three failed IUI cycles and a high DFI, she counselled the couple for ICSI, in which a single sperm is injected directly into each egg. The woman conceived after the first embryo transfer and was five months pregnant at the time of the masterclass. Beyond the result, Dr. Singh noted, the test gave the couple an objective reason to move on from IUI, which made the decision easier for them.

Medical treatment for men: a small but specific role

Medical treatment helps only a minority of infertile men, and the endocrine results decide who. Dr. Singh's algorithm starts from total testosterone. If it is normal, drugs have little to offer. If it is below 300 ng/dL, the next step is FSH and LH:

1. Low testosterone with low FSH and LH: hypogonadotropic hypogonadism. The pituitary is not driving the testes. The cause may be congenital or acquired, and these men respond to gonadotropins: human chorionic gonadotropin (hCG) combined with FSH, or FSH combined with LH. This is the group in which even azoospermia can be treated medically.
2. Low testosterone with normal or raised FSH and LH: calculate the testosterone to oestradiol ratio. Normally it is above 10.

• Ratio below 10: aromatase hyperactivity. Aromatase is the enzyme in fat tissue that converts testosterone into oestradiol. Men with central (truncal) obesity convert more testosterone into oestrogen, which impairs sperm production. Aromatase inhibitors such as anastrozole, letrozole or testolactone are useful here.
• Ratio above 10: primary testicular failure (hypergonadotropic hypogonadism). Men with severe oligospermia or non-obstructive azoospermia in this group are candidates for IVF with ICSI. A few who are not ready for IVF may benefit from anti-oestrogen drugs; meta-analyses have found clomiphene useful in this setting.

Antioxidants: widely prescribed, selectively indicated

Dr. Singh observed that almost every physician prescribes at least one multivitamin or antioxidant combination to an infertile man, usually without measuring oxidative stress. Antioxidants are genuinely indicated when oxidative stress is present. Without ROS or DFI testing, a careful history identifies the likely cases: varicocele, infection, smoking, medication or chronic systemic illness, and a high concentration of white cells in the semen. Many combinations are available, containing ingredients such as zinc and ascorbic acid, and no formulation has been proven superior to another.

Surgery for male infertility

Surgery has a defined place:

• Varicocele repair when the varicocele is of a severe degree and associated with abnormal semen, provided the female partner's evaluation is normal. Costs at partner hospitals are summarised on the varicocelectomy cost page.
• Relief of obstruction, for example removal of a midline cyst in the lower genital tract.
• Surgical sperm retrieval. When the testes are failing, sperm can be retrieved directly from testicular tissue by testicular sperm extraction (TESE), or by micro-TESE, which uses an operating microscope to find the small areas of tissue still producing sperm. These sperm are used for ICSI.

Two common clinical problems

Oligospermia

For a low sperm count, Dr. Singh's approach has four steps. If the count is very low, rule out genetic causes with a karyotype and Y-chromosome microdeletion testing. Correct modifiable factors such as smoking and alcohol, and give antioxidants. Repeat the semen analysis after about three months. If the count improves and genetic tests are normal, medical treatment with IUI is reasonable; if it does not improve, move to assisted reproduction.

Sperm in the urine: retrograde ejaculation

Sperm found in the urine indicate retrograde ejaculation, in which semen flows backwards into the bladder instead of forwards. It follows pelvic, perineal or urological surgery, and occurs rarely in diabetes. Natural conception is unlikely, but the sperm can still be used. The man takes an alkalinising agent; after ejaculation he passes urine, which is collected and centrifuged to separate the sperm. Depending on the concentration, those sperm are used for IUI or for IVF with ICSI. Any infection of the epididymis is treated with antibiotics first.

Where the male evaluation leads

Most of the decisions above end in one of three places: medical treatment, IUI with prepared sperm, or IVF with ICSI or its refinement, PICSI, which selects sperm with better DNA integrity. Those treatments are described in IUI, IVF and ICSI: the assisted reproduction ladder. The full audience questions on retrograde ejaculation and oligospermia are in the Live Discussion from this masterclass.