DEVELOPMENT OF SEMEN QUALITY IN MALE PARTNERS OF INFERTILE COUPLES IN THE REPUBLIC OF MOLDOVA

methods. purpose of the study is to analyze the regional tendencies of the semen quality in male partners of couples facing infertility. A retrospective study of 4625 patients subject to semen analysis between 2012-2018 was conducted. All semen samples were collected after a recommended period of sexual abstinence of three to five days. The spermiogram analysis was performed by the computerized method according to WHO guidelines for Human Semen analysis, 2010. Results. Of the total number of 4625 men examined, 1861 (40.2%) presented normal values of semen – normozoospermia, and 2764 (59.8%) showed abnormal semen parameters. Asthenozoospermia was the most common abnormality profile recorded in 1394 (30.2%) men, followed by oligoasthenozoospermia diagnosed in 973 men (21.0%). Azoospermia was found in 200 men with an estimated prevalence of 4.3%. In 113 men examined, oligozoospermia was

Infertility affects an estimated rate of 15% of couples of reproductive age worldwide, and in about half of these cases the male factor is involved (1).
The causes of infertility can be divided into four broad categories: 1) female factor; 2) male factor; 3) couple factor -due to cumulative female and male infertility; 4) idiopathic infertility, unexplained. The exact percentage for each of these categories is difficult to determine; however, it is generally reported that in about 40% of cases infertility is due to female cause, in 40% -male cause, and in 20% -anomalies detected in both partners (1,2). Thus, the examination of the male partner is as important as the female one for the assessment of couple's fertility. Medical history and physical examination are standard assessments for all men, including semen analysis.
The spermiogram evaluation is relevant for the appreciation of the functional status of the seminiferous tubules, epididymis, and accessory sex glands. The prognostic value of semen characteristics, such as sperm concentration, percentage of motility, and morphology represents the first line of examination in the diagnosis of male infertility (3). Semen analysis may not always be an optimal diagnostic tool, but it still remains the basic clinical tool for the evaluation of male fertility potential (4). Important treatment decisions in male infertility are largely based on spermiogram results. There-fore, it is essential that the human semen analysis be performed according to the updated requirements of the World Health Organization (WHO), 2010 (5). In recent years, the European Society for Human Reproduction and Embryo-logy (ESHRE), in collaboration with the WHO, have developed a program to improve laboratory standardization in terms of sperm sample diagnosis and assessment criteria (6).

MATERIAL AND METHODS
The purpose of the study is to analyze the regional tendencies of semen quality in male partners of couples facing infertility.
The study presents a retrospective evaluation of 4 625 patients in the Republic of Moldova subject to semen analysis during 2012-2018. All semen samples were collected in laboratory conditions after a recommended period of sexual abstinence of three to five days. Each sample was incubated at 37 o C and analyzed within an hour. The spermiogram analysis was performed by the computerized method on the automated analyzer SQA IIC-P (Medical Electronic Systems, USA). Semen analysis was performed according to the WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edition, 2010 (tab. 1). All patients are part of infertile couples who made appointments for doctor`s consultation in the Repromed Center. Interpreting the results, the spermiogram diagnosis was made according to the descriptive terminology of the same WHO guidelines as follows: -normozoospermia: total number/percentage of sperm with progressive mobility and normal morphology, being of equal value or above the reference values; -oligozoospermia: total number of sperm/sperm concentration below lower reference limit; -asthenozoospermia: sperm motility below 40% or rapid progressive sperm motility <32%; -teratozoospermia: percentage of normal sperm below 4%; -oligoasthenozoospermia: low concentration and low percentage of progressively motile sperm; -oligoteratozoospermia: low total number of sperm and low percentage of normal forms; -asthenoteratozoospermia: percentage of motile sperm and normal sperm below low reference limit; -oligoasthenoteratozoospermia: low total number of sperm/low percentage of motile sperm and normal forms; -cryptozoospermia: very low spermatozoa concentration in ejaculate ≤1 million/mL; -hypospermia: semen volume < 1,5 mL; -hyperspermia: semen volume >1,5 mL; -leukospermia/pyospermia: presence of leukocytes in ejaculate above reference limit; -hematospermia: presence of blood in ejaculate; -necrozoospermia: low percentage of live and high percentage of immotile sperm; -aspermia: complete lack of semen with ejaculation; -azoospermia: absence of spermatozoa in the sediment of a centrifuged semen sample.
Normozoospermia was considered according to the following WHO criteria: sperm concentration ≥1.5 mln/mL, total number of sperm cells ≥39 mln, progressive sperm motility ≥32% and morphology ≥4%

DISCUSSIONS
The study shows that during 2012-2018, the abnormal semen quality was found in approximately 59.8% of male partners of couples facing infertility (tab. 2). A high incidence of spermatogenic disorders is also found in other studies (7,8,9) According to our data, this percentage increased from 50% in 2012 to 66.3% in 2018 (tab. 2).
This represents a significant increase in spermatogenesis abnormalities, although the WHO introduced lower baseline values in 2010. Thus, an alarming phenomenon of decreased male fertility can be observed in fertility clinics in the Republic of Moldova, which could be correlated with sperm decline described in the literature. This has been illustrated by several studies from multiple world regions that argue that men's reproductive health has been in rapid decline in the recent years (10,11).
In the present study we observe that sperm concentration in the semen and healthy sperm count have decreased considerably. If in 2012 and 2013 the frequency of normozoospermia was 50.9%, over the following years a decrease in the normal values of spermatogenesis could be observed, accounting for 33.7% in 2018 ( fig. 2).
The most common profile of abnormality in our study was asthenozoospermia recorded in 1394 men with a frequency of 30.2% ( fig. 1). It is very noticeable that the abnormal frequency of sperm motility increased from 2012 to 2018. Thus, in 2012 the frequency of asthenozoospermia was 21.7%, in 2014 -28.6% it increased, in 2015 -28.8%, 2016 -32.7%, in 2017 -35.5%. In 2018 there was a slight decrease of asthenozoospermia -32.6%, most probably due to a lower number of appointments of couples with infertility compared to the previous year ( fig. 3). According to literature data, this abnormality is found in 40% of men, affecting their fertility. The role of socio-psycho-behavioral factors in the development of this abnormality has been demonstrated. Psychological stress, smoking and alcohol are modifiable risk factors for the number of motile sperm (12). Also, sperm motility depends on its specific structures such as microtubules, outer dense fibers and mitochondria that provide energy for sperm movements (13).
The second cause of spermatogenesis disorder identified in our study was oligoastenozoospermia with a frequency of 21.0%. Oligoastenozoospermia is a combination of reduced sperm motility and low sperm count. According to bibliographic sources it is the most common cause of male infertility (14). The causes of this disorder are heterogeneous, such as cryptorhidia, varicocele, chronic infections, hormonal causes, psychoemotional causes, metabolic causes, etc. In 20% its etiology and pathogenesis are not fully elucidated and may be associated with specific gene abnormalities (13).
The frequency of azoospermia in the current study accounts for 4.3%. According to bibliographic sources azoospermia is found in 8% of infertile men and in 1% of the male population (15). In the case of lack of sperm or an extremely small number, a genetic cause may be identified in about 21-29% (16). In this context, cytogenetic tests for karyotype analysis as well as molecular genetic evaluation of Y-chromosome microdeletions analysis and mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene are fully justified and important to exclude a possible cause of genetic origin. Abnormal genotype may be present in up to 12% of azoospermic men and 4% of oligospermic men. Cystic fibrosis screening is recommended for azoospermia if it is due to congenital bilateral absence of the vas deferens (CBAVD). Optional Y-chromosome microdeletion screening can be carried out if sperm count is <5 million/mL.

CONCLUSION
1. Our results clearly show that semen quality in the population of men in couples with infertility in the Republic of Moldova decreased from 2012 to 2018. As many authors suggest, we also believe that environmental and lifestyle factors have negatively affected the quality of semen development. The contribution of genetic factors cannot be excluded either.
2. Therefore, the analysis of the regional tendencies of semen quality is necessary and can be considered an indirect factor in assessing the tendencies in male infertility.