Infertility is a disease of the male or female reproductive system defined by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse.^[1] As per the WHO estimates suggest that between 48 million couples ( 1 in 7 couples ) and 186 million individuals live with infertility globally and up to 50% of these couples, male infertility plays at least a partial role.^[2,3] 40% of infertility cases were related to men, 40% of women and 20% of both sexes.^[4]

The diagnostic approach to a case of male infertility includes a detailed clinical history, physical  examination and laboratory investigations like semen analysis, hormonal evaluations etc.  and invasive tests like testicular biopsy (or) FNAC (Fine Needle Aspiration Cytology). FNAC has become one of the important methods of investigations in cases of azoospermia and oligospermia. Though testicular biopsy provides some useful information regarding spermatogenesis and basement membrane status of seminiferous tubules, it has its own complications like hematoma, fibrosis , scarring and sampling only a small volume of tissue. FNAC on the other hand is reliable, quick, easy, less invasive and associated with no or minimal complications and can be performed without anaesthesia multiple times.  Studies have shown a good correlation between FNA and biopsy findings and abnormal findings in FNA can be followed up by biopsy. This study evaluates the value of percentage of cell counts and indices such as spermatic index, sertoli cell index and sperm-sertoli cell index; correlate the findings with the histopathological findings for accessing the accuracy of the cytological finding.

The study was conducted between August 2017 and November 2019 in the Department of  Pathology/Urology, SCB Medical College,Cuttack after institutional clearance. The absolute indications, relative indications and contra indications in testicular biopsy/FNAC are Azoospermi/Oligozoospermia, Varicocele/cryptorchidism/Chronic infection and  Severe anaemia/Unco-operative patients respectively.^[5]

The study was done after obtaining informed consent in 48 patients in whom three consecutive semen analyses showed oligospermia or azoospermia. All semen analyses were done only after a period of abstinence of at least 4 days. Patients were then subjected to a FNA of the testes for cytological evaluation and testicular biopsy was done for histopathological correlation. Then the results sperm count in semen analysis are given as either normal (sperm count >15 million) or oligospermic (sperm count <15 million) or azoospermic (no sperms seen).

FNAC of testis of 48 patients was done from middle position of both the testis opposite to epididymis with single pass & aspiration was done without anaesthesia. Then the air-dried smears were stained with May-Grunwald-Giemsa(MGM) and alcohol-fixed smears were stained with Papanicolaou(PAP)  and Haematoxylin& Eosin (H & E).

Biopsy of the testis with anaesthesia was done by 1 to 1.5 cm incision made on the convexity of the centre of anterior portion of testes. Excised specimen fixed in Bouin’s fluid, specimen processed and stained with H & E for HP examination. After securing homeostasis, Tunica albuginea, Tunica Vaginalis and skin with subcutis were closed in layers with 3/0 vicryl sutures.^[6]

Cytological Grading: Cytological smears were  graded from A to D as per Table-1 by counting 100 consecutive spermatogenic and sertoli cells in a well spread area with good cellularity in a random manner using light microscope. ^[7]

Cytological diagnosis: Based on various proportions of the different cell types, the smears were categorized into five groups (Table-2).^[8]

Cell Indices:100 consecutive spermatogenic and sertoli cells were counted in a well spread portion of the smear and the percentage of spermatozoa per 100 spermatogenic cells (SI- Spermatic index), the number of sertoli cells per 100 spermatogenic  cells (SEI- Sertoli cell index) and number of spermatozoa per 100 sertoli cells(SPSEI- Sperm sertolicell index)were calculated.^[9]  The mean value of these indices in each category of the cytological diagnosis is then taken and compared with different categories of cytological diagnosis

In normal spermatogenesis: SI and SPSEI will be higher than SEI. In hypospermatogenesis: SI and SPSEI will be lower than in normal spermatogenesis and SEI will be higher than in normal spermatogenesis and maturation arrest. In maturation arrest: SI and SPSEI will be zero and SEI will be higher than in normal spermatogenesis. In SCOS: SI will be zero and SEI & SPSEI cannot be calculated as there are no sertoli cell.

HISTOLOGICAL DIAGNOSIS: In the histopathology, the predominant pattern was taken into account and classified into the following five patterns.^{[ 6]}

1. Normal spermatogenesis: Germ cells in all stages of spermatogenesis are seen within the seminiferous tubules.
2. Hypospermatogenesis: A reduction in the number of all germinal elements, including the late spermatids/spermatozoa is present.
3. Maturation Arrest: Histological examination reveals spermatogenesis proceeding normally through a specific stage at which point no further maturation of germ cells is identified. The arrest may occur at the primary spermatocyte, secondary spermatocyte or early spermatid stage.
4. Germ Cell Aplasia: Testicular histology reveals seminiferous tubules lined by sertoli cells with a complete absence of all germ cells.
5. Tubular hyalinization: Hyalinization of the tubules is associated with a loss of germinal epithelium, obliteration of the lumens and fibrosis of the interstitium. Sertoli cells may or may not be present. Leydig cells may be absent or decreased in number within the sclerotic interstitium.

Different cell index in Testicular FNAC smear in different histological categories.^[10]

JOHNSEN SCORING: 100 consecutive seminiferous tubules were counted and the mean was taken and compared with histological report.^[11]

Score 10 - Complete Spermatogenesis and perfect tubules

Score 9 - Many spermatozoa present but disorganized spermatogenesis

Score 8 - Only a few spermatozoa present

Score 7 – No spermatozoa but many spermatids present

Score 6 - No spermatozoa, only a few spermatids present

Score 5 - No spermatozoa or spermatids present, but many spermatocytes present

Score 4 - Only a few spermatocytes present

Score 3 - Spermatogonia only

Score 2 - No germ cells, sertoli cells only

Score 1 - No germ cells or sertoli cells

The mean Johnson Score of normal testis is 9.39±0.24

Statistical analysis was performed by using IBM-Statistical Product & Service Solutions (SPSS) software, Version 20.

This study was done on 48 cases. The age group ranged from 20 to 40 years (Table-4 ) with a meanage of 33.6 yrs.

The duration of infertility ranged from 2 years to 14 years (Table-5)with mean duration of 6.33 years.

In semen analysis, the sperm count ranged from azoospermiatooligospermia (<15million). Azoospermia is correlated with maturation arrest, germ cell aplasia and tubular hyalinization. Oligospermia is correlated with hypo-spermatogenesis and normal-spermatogenesis (Table -6)

Out of 48 cases, 38 cases (79.17%) were azoospermic. Out of this 26 cases (68%) correlated with HP diagnosis and 25 cases (66%) correlated with cytological diagnosis. Discordance of 12 cases in HPE and 13 cases in cytology was found to be due toobstructive causes. Out of 48 cases, 10 cases (22.86%) were oligospermic; out of which 6 cases (60%) correlated with HP diagnosis and 8 cases (80%) correlated with cytological diagnosis.

Cytological smears were categorised on the basis of cell types into 5 groups, graded from A to D based on cellularity and correlated with the cytological diagnosis (Table-7).^[7-8,12-13]

The most frequent diagnosis on cytological report was Maturation arrest with 19 cases followed by Normal spermatogenesis in 11 cases,  Hypospermatogenesis & SCOS were 8 cases each and Testicular atrophy in 2 cases. The common cytological grades in the descending order of frequencies were B, A1,A2 & C and D.

The mean value of cell indices (SI & SEI) in each category of the cytological diagnosis is tabulated Table - 8.[^8,9]

Progressive decreasing values of SI were seen in normal spermatogenesis, hypospermatogenesis and maturation arrest. SEI was found to be the most important index in distinguishing hypospermatogenesis from normal spermatogenesis since SEI was found to be elevated in hypospermatogenesis. Maturation arrest was distinguished from hypospermatogenesis by the SI which was severely decreased with absent spermatozoa. In SCOS, SI and SPSEI were zero.

In histological classification, the predominant pattern was taken into account and presented in Table 9.^[6]

Johnsen scoring: 100 consecutive seminiferous tubules were counted and the mean was taken and comparedwith histological diagnosis (Table –10).^[6,11]

Most of the cases scored between 7-3 which means maturation arrest. Scores 9 & 10 correlated well with Normal spermatogenesis, score 2 & 1 with Germ Cell Aplasia and Tubular hyalinization. There were discordance in one case, where score 7 was reported as Hypospermatogenesis.

Considering histopathology as the gold standard for definitive diagnosis of any lesion, of the 48 cases of our study, 47 cases correlated well with FNAC (Table-11). The overall percentage of correlation with respect to HPE was 97.91%. Of 20 casesdiagnosed as Maturation arrest in HPE, 19 cases were diagnosed the same in FNAC. Remaining 1 case was diagnosed as Hypospermatogenesis since the smears contained few mature spermatozoa.

The p-value of the study is 0.0325 which is ≤ 0.05 is considered significant.

Pictomicrograh of various patterns as per FNAC & HPE are illustrated in Fig-1 to Fig-5.

Fig-1: a) Cytosmear showing normal spermatogenesis (MGM x400) & b) HP showing normal spermatogenesis (H&E x400)

Fig-2: a) Cytosmear  showing   hypospermatogenesis  (MGM x400) & b) HP section showing hypospermatogenesis  (H&E x400)

Fig-3: a) Cytosmear showing maturation arrest (MGM x400) & b) HP section showing maturation arrest (H&E x400)

Fig-4: a) Cytosmear  showingsertoli cells only (MGM x400) & b) HP section  showing  sertoli cells only (H&E x400)

Fig-5: a) Cytosmear showing leydig cell of testicular atrophy (MGM x400) & b) HP showing tubular hyalinization  (H&E x400)

Open testicular biopsy has remained the cornerstone in the diagnosis of maleinfertility for decades. Testicular FNAC has picked up in recent years following Papic et al, Foresta et al,Obrant et al, and Schencket et al who characterized different cell types in cytological smears and demonstrated good correction ofcytological diagnosis with histological categories.^[12-15] In current era, microassisted fertilization techniques are of great help for infertile couples as nowadays the only requirement in these techniques is a viable sperm and ovum. Neither quality nor degree of motility is essential.^[16] FNAC  which is minimally invasive, a report of presence/absence of sperm is also adequate. In cases of hypospermatogenesis and maturation arrest, these patients may be helped to some extent by hormonal therapy. FNAC serves the purpose with minimum side effects whereas biopsy may result in fibrosis which hampers the process of sperm extraction for Intra Cytoplasmic Sperm Injection(ICSI).

This study has been done in 48 infertile with mean age of 33.6 years. Out of these 48 cases, 38 cases were found to be azoospermic and 10 cases found to be oligospermic. Out of the 38 azoospermic cases; 12 cases in HPE and 13 cases in FNAC were found to have mature sperms.  This discordance was found to be due to obstructive causes.

The mean age of infertility of various studies and current study is tabulated in Table-12.^[7-8,13]

The duration of infertility was found to be 2-10 years according to Mona et al, 2 years according to Foresta et al and 6.33 years in the current study.^[13,17]

Cytomorphological patterns of various studies as compared with current study (Table-13).^[6,18-21]

Table-14  compares the current  study with the study by  Dajani et al  w.r.t.  grading of cytosmears.^[7]

Patterns of testicular histopathology ranked according to the Johnsen scoring system in the current study is compared with the study by Mona et al in Table-15.^[17]

Table-16 gives Correlation between FNAC and biopsy of the previous and he current studies.^[20,22-28]

Most of the available references show an accuracy rate of >84% and the current study shows % agreement of 97.9%. Discordance was observed in 1 out of 48 cases (2.1%), which was reported as maturation arrest in biopsy was found to have mature spermatozoa (hypospermatogenesis) in FNAC.

Most of the studies performed FNA under general anaesthesia (cord block). Study by Verma AK et al and the current study was performed FNA without anaesthetia and was well tolerated by the patients.^[18]

There are also some limitations as FNAC is unable toprovide architectural information of testes about thickness of tubular basement membrane, tubular diameter or status of interstitial tissue. Testicular disorders leading to azoospermia such as atrophy, fibrosis and Leydig cell hyperplasia can be diagnosed better by histology.  Patients may complain of prolonged pain but this can be relieved by scrotal support and analgesics. Neurogenic shock has been reported in patients who failed to rest after the procedure.

FNA is simple, quick, less expensive, less invasive and minimally traumatic with less number of complications in comparisons to biopsy. It can be performed without anaesthesia and multiple specimens can be taken safely. Micro-assisted fertilization techniques are of great help for infertile couples as nowadays the only requirement in these techniques is a viable sperm and ovum. Therefore in cytological smears a report of presence orabsence of sperm is also adequate. However, in testicular biopsy of male infertility, cytology could beconsidered as gold standard as histologically negative cases show sperm in cytology. Sperm extraction for ICSI could be successfully done based on cytology report rather than histology report. The current study showed a correlation between biopsy and FNAC as high as 97.9% and the p value of the study was 0.0325.

Conflicts of interest: Nil

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