Part four of the expert cycle: “Cytogenetic testing in the assessment of female fertility” deals with the assessment of the fertility of both partners by means of cytogenetic testing. The proper performance of diagnosis followed by a specialist genetic advice for the couple significantly increases the chance of normal development of pregnancy in couples with abnormal karyotype.

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Cytogenetic testing in the assessment of female fertility

According to WHO, infertility is an inability to conceive after one year of regular unprotected sexual intercourse [1]. Approximately 10% to 15% of pregnancies confirmed with the clinical diagnosis made in the 1st trimester end in spontaneous abortion. According to WHO, at least 3 consecutive miscarriages are defined as habitual abortions, but already 2 miscarriages are the indication for the cytogenetic testing of both partners. This problem affects 1% to 2% of all couples of reproductive age [2]. It is estimated that among couples who experience recurrent miscarriages, on average 3.0% to 6.0% of partners carry chromosomal structural aberrations which occur twice more often in female population than in men [3, 11]. While before the era of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), the determination of the type of chromosome anomaly did not play any significant role, now, in the period of developing assisted reproductive technologies, cytogenetic testing for the detection of chromosomal abnormalities has gained importance. Detecting a chromosome aberration is important when diagnosing infertility, choosing a treatment, estimating the risk of passing on the defect to future children and exercising appropriate oversight over a pregnant woman [4]. The cause of infertility without treatment affects further prognosis, but it is obvious that it can’t be determined without appropriate tests [5]. The diagnosis of unexplained infertility is made when all standard elements of the assessment bring normal results. The minimum requirements for such diagnosis are: a normal result of semen analysis, objective evidence of ovulation, normal uterine cavity and patency of both fallopian tubes. The next stage of diagnosis is cytogenetic testing, since the studies conducted by Mau Holzmann in 2005 demonstrated that in couples with the history of multiple miscarriages, there was an increased incidence of balanced translocations or other chromosomal abnormalities, which did not affect phenotype, but at the same time might be lethal for the zygote with unbalanced karyotype [6]. Thus, a correlation between the number of miscarriages and the incidence of chromosomal abnormalities was noticed [3].

It has been long debated whether the analysis of karyotype in women can be used as the routine diagnostic test for infertile couples [7]. As chromosome aberrations can be the cause of infertility both in women and in men, most authors notice the need to carry out cytogenetic screening tests in both partners who decide to use the methods of assisted reproductive technology [1, 2].

The most common clinical situations where it is indicated to determine the karyotype of the patient and her partner in the departments and outpatient clinics of gynecology, gynecological endocrinology and infertility treatment include:

  • azoospermia or major oligozoospermia in semen analysis (Klinefelter’s syndrome),
  • developmental defects in a child who died after delivery, or in a dead fetus (autosomal trisomies, unbalanced translocation, deletion),
  • a chromosome aberration detected in a prenatal test or an atypical morphological variant of a chromosome detected in a patient’s fetus,
  • infertility of unknown etiology,
  • abnormal structure of reproductive organs, intersexuality (Turner’s syndrome, XY women),
  • presence of a structural aberration in the family – in this case, it is necessary to examine other members of the family in order to determine the risk of aberrations in the progeny in this family.

Furthermore, karyotype analysis is indicated in women with:

  • primary or secondary amenorrhea of undetermined etiology or with premature menopause (suspected Turner’s syndrome, women with an extra Y chromosome),
  • significant growth disorders of unknown etiology (Turner’s syndrome, Klinefelter’s syndrome, 47, XXY),
  • presence of a recessive disease associated with the X chromosome, two (or more) spontaneous abortions in the 1st trimester of pregnancy (referral after the exclusion of other, more common causes) or recurrent miscarriages also in the later period of pregnancy [8].

Another important indication for cytogenetic testing is the diagnostic procedure before using the methods of assisted reproductive technology [9]. An abnormal result of the cytogenetic test should be given to the patient in the framework of the genetic advice. Its interpretation requires not only the knowledge about the chromosome syndromes and aberrations but also a good knowledge of the technique the test was performed with and its limitations.

The area of application of the cytogenetic tests which aim at detecting chromosomal abnormalities is limited due to their resolution. In a conventional high-resolution cytogenetic test, it is possible to visualize aberrations at the level of one band (with the length of up to ca. 5 Mbp, i.e. 5,000,000 base pars, while when using the HR-CGH (high resolution comparative genome hybridization) it is possible to visualize micro-aberrations, that is deletions or duplications covering the chromosome sections ca 3 Mbp long. Thanks to the microarray technology, the test resolution can amount to as many as 1.4 Kbp. Currently, the whole genome array CGH tests are used, but also arrays for the examination of changes in individual chromosomes.

Microarrays contain the sets of desired high-resolution cloned DNA sequences or oligonucleotides, which allows to detect polymorphisms in a DNA sequence, the so-called SNPs (single nucleotide polymorphisms).

Molecular methods allow to detect changes as small as the mutations in single nucleotides. In some cases, where the occurrence of a given syndrome might be determined by the presence of micro-aberrations or genetic mutations, complete genetic diagnosis may consist of both the conventional cytogenetic testing and molecular cytogenetic testing (e.g. FISH). In the case of genetic diseases where the background of their occurrence is not a chromosome aberration but mutations within one or several genes, these changes cannot be detected with cytogenetic methods. In such cases, various methods of molecular biology are applied. The knowledge of the etiology of a given genetic syndrome facilitates the planning of the diagnostic process.

Taking into account the development of the modern methods of molecular biology, cytogenetic techniques can be divided into conventional and molecular ones:

  • Conventional cytogenetics is based on the methods of chromosome band analysis.
  • Molecular cytogenetics uses the techniques of molecular biology to test for chromosome normality.

Molecular cytogenetics is mostly based on the method of FISH – fluorescent in situ hybridization- which uses the so-called probes for the detection of specific chromosome aberrations. These probes detect specific DNA sequences. They can be specific for centromeres, telomeres, specified bands or even whole chromosomes. Their main advantage is their ability to detect aberrations in interphase nuclei without the need for in vitro culture, but already before the test a probe detecting the specified aberration has to be chosen; at the same time, the use of conventional cytogenetics allows the numerical analysis of all chromosomes and the detection of an a priori unsuspected aberration.

Molecular cytogenetics complements tests carried out with the use of the methods of conventional cytogenetics which forms the foundation of the workshop. Molecular cytogenetics, however, is the source of tests for specified aberrations or of solutions to diagnostic problems.

Fluorescent in situ hybridization (FISH)

The basis for its operation is the principle of base complementarity in DNA chains. Each chromosome is built of two chromatids, and each chromatid is built of one DNA molecule, that is one double DNA helix and proteins.

It is possible to carry out the denaturation of chromosomal DNA. A single-stranded DNA will strive to find the complementary DNA strand and to attach itself to it, i.e. hybridize. In the FISH test, a short section of DNA with a known sequence (a DNA probe) hybridizes to a DNA strand on a chromosome. In order to make the observation of the very short DNA probes possible, they are marked with a fluorescent particle or any other particle which is detected with the use of antibodies marked by a fluorescent material. Thus the name of fluorescent hybridization; its result is observed in a fluorescence microscope as fluorescent signals on chromosomes. FISH is carried out on preparations which contain DNA in the form of chromosomes. This test allows to detect specific DNA sequences on chromosomes, individual cells and tissues. The classical FISH technique uses one to three probes with different marking. If a patient has a translocation, a signal from the probe on the chromosome other than the normal location of a given probe will be observed. In this way, using the appropriate combination of probes, it is possible to detect sets of microdeletions, confirm translocations, insertions and other aberrations and to solve the diagnostic problems encountered by a cytogenetician during the interpretation of chromosomal changes in a conventional cytogenetic test.

Applications of the classical FISH technique:

  • examination of the normality of the chromosome structure,
  • diagnosis of aberrations in the chromosome structure,
  • precise determination of the chromosomal breakpoints in the diagnosed aberrations in the chromosome structure,
  • detection of small deletions in the case of apparently cytogenetically balanced de novo translocations,
  • identification of subtelomeric aberrations,
  • cytogenetic diagnosis of cancer cells,
  • solving diagnostic problems resulting from the band analysis,
  • determination of the occurrence of certain morphological variants of chromosomes.

Interphase cytogenetics

A technique which allows to examine chromosomes in an interphase nucleus with the use of molecular methods. Detection of the specified fragments of DNA on the interphase nucleus significantly facilitates and accelerates certain cytogenetic tests as it is not necessary to obtain dividing cells in the cell culture for chromosome examination. Interphase cytogenetics is used mainly in prenatal, oncological and hemato-oncological tests and in preimplantation testing.

Multicolor FISH

The operating principle of this technique is identical to the fluorescent in situ hybridization. This test is actually a variant of FISH in which different DNA probes are used. It consists in the simultaneous staining of all chromosomes with different colors. Each pair of homologous chromosomes is stained with different color. This technique is useful in the analysis of complex aberrations, especially when several chromosomes take part in the translocation and when it is hard to differentiate the origin of fragments of chromosomes with multiple damages.

Comparative genomic hybridization

The CGH technique is also a one of modifications of the FISH method. During the CGH test, hybridization in situ is carried out on the preparation which contains normal metaphases. As the probe, marked DNA is used:

  • a patient’s genomic DNA stained green,
  • marked genomic reference DNA stained red.

The greatest advantage of this technique is that it can be used on tissues from which no dividing cells could be obtained, and in spite of this it is possible to identify the unbalanced aberrations of every chromosome. The disadvantages of this technique include the lack of the possibility to detect balanced aberrations like translocations or inversions and rather limited possibilities of examining telomeric aberrations and mosaicism.

Due to the developments in molecular cytogenetics, at present, plenty of molecular techniques which can be useful in DNA testing are used for testing for chromosome normality. This area of cytogenetics interlocks closely with molecular genetics. The use of these methods, similarly to FISH, allows detecting increasingly smaller deletions and duplications or detecting aneuploidies in tissue cells without the procedure of culture, just as it happens in the GCH technique.


The array-CGH technique is a modern method where a patient’s genomic DNA is used as the probe, and a reference DNA which is used for comparison. In this technique, the hybridization of genomic probes is carried out on an array containing DNA clones or oligonucleotides. Undoubtedly, this technique is at present the most precise (it has the highest resolution) method of testing for chromosome aberrations.


The MLPA (multiplex ligation-dependent probe amplification) technique, contrary to the techniques described earlier herein, is not based on in situ hybridization but it combines the oligonucleotide hybridization to patient’s DNA with the PCR technique. A huge advantage of this technique is the possibility of using as many as 45 probes in one reaction and a limited amount of DNA (20ng-approximately 3,300 cells or 1 mL of amniotic fluid) which is necessary for the reaction. The MLPA probes can recognize even the differences in a DNA sequence in the form of a single nucleotide mutation which allows to use this technique not only in testing for chromosome aberrations but also for DNA molecular testing. This technique, however, has its limitations, namely it does not detect balanced chromosome aberrations, and the type of detected aberrations depends on the set used.

After obtaining the test result, one has to assess whether a given normal result actually makes further diagnosis unnecessary; while in the case of a pathological result, it has to be interpreted properly, and the patient and his or her family has to be given an appropriate genetic advice [10]. It is worth mentioning here that couples with recurrent miscarriages and confirmed chromosomal abnormalities have the chance for the birth of a healthy child but they should be informed about the risk of another miscarriage [3].

Studies demonstrate that recurrent miscarriages resulting from the anomalies in the karyotype of the potential parents are more often related to balanced structural aberrations carried by women than by men [1, 3]. Male partners of an infertile couple with indications to IVF procedure show the incidence of chromosomal abnormalities on the level of 1.0%, while patients with indications to ICSI show karyotype anomalies on the level of 4.6%. What is interesting is that women with indications to IVF show seven-fold higher incidence of chromosomal abnormalities (4.0%), while those with indications to ICSI have the incidence of karyotype anomalies on the level of 5.0% [9].

Cytogenetic tests carried out in women as a part of routine tests preceding the application of assisted reproductive techniques show the following abnormalities:

  • 47, XXX syndrome (only 0.1% of patients subjected to testing)
  • The Turner’s syndrome (45,X) (45,X/46,XX or 45,X/47,XXX/46,XX, etc.), which is the most common chromosome aberration associated with female infertility and ovarian failure, in tests is more often detected as gonadal mosaicism (45,X/46,XX or 45,X/47,XXX/46,XX, etc.) than monosomy (45,X). Such result is sought in connection with a specific phenotype (low height, absence of secondary sex characteristics, amenorrhea) in the case of monosomy which speeds up the patient’s diagnosis.
  • Structural aberrations of the X chromosome: i(Xq) and del(Xp) which are the most common structural chromosome aberration with the concurrent phenotype characteristic for the Turner’s syndrome. The anomaly consists in the presence of an isochromosome in which the long arms of the X chromosome join each other. It is estimated that 15% of the karyotyped patients with the Turner’s syndrome have i(Xq).
  • The X-autosome translocations usually emerging de novo. A patient’s phenotype and fertility depend on many factors, including the point where the chromosome breaks. This translocation may be inactivated when the translocation chromosome preserves its inactivation center [12].
  • Robertsonian translocations found in a small (0.2%) percentage of women subjected to tests do not play any major role in the pathogenesis of female infertility.
  • Reciprocal autosomal translocations. Their incidence in women and in men does not differ significantly. The aberrations of this type occurred in 1.0% of women before IVF and in 0.7% of women before ICSI.
  • Complex chromosomal rearrangements (CCR) are structural chromosomal abnormalities concerning at least three chromosomes with three or more breakpoints. Detailed data on the incidence in overall population are not available but it is known that in majority the cases of this abnormality which have been described in the literature so far have occurred in women. They can emerge de novo or be inherited from a mother.
  • Inversions with the incidence of 0.01-0.07% for the pericentric type and 0.01-0.05% for the paracentric type. The carriers of this aberration are more often women. Its incidence in women subjected to tests was 0.4% before IVF and 0.3% before ICSI.

The detection of the abnormalities mentioned above may have major impact on the course of planned treatment (IVF or ICSI) and on its successful outcome [9]. For this reason, it is important to carry out cytogenetic testing for the purpose of assessing the fertility of both partners. The proper performance of diagnosis followed by a specialist genetic advice given to the couple significantly increases the chance of normal development of pregnancy in couples with abnormal karyotype [3].

Authors of article: Dagmara Krajewska, prof. dr hab. Krzysztof Łukaszuk, Kierownik Klinik Leczenia Niepłodności INVICTA

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K., Wyniki badań cytogenetycznych i molekularnych u 35 par z niepłodnością pierwotną, Wiadomości Lekarskie 2006, LIX, 1-2
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107 par z regionu kujawsko-pomorskiego z nawracającymi poronieniami samoistnymi, Wiadomości lekarskie 2005, LVIII, 11-12
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5. Leon Sperof, M.D., Marc A. Fritz, MD. Clinical Gynaecologic Endocrinology and Infertility, Seventh edition, 1179
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analysis mandatory in the initial investigation of normovulatory women seeking infertility treatment?, Human Reproduction 20 (10: 2899-903, 2005
8. I. Srebrniak M., Tomaszewska A., Badania cytogenetyczne w praktyce klinicznej, PZWL, 2008, 50


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Published: 5 November 2015 Updated: 4 April 2017