A study on glycogen content of endometrial glands in infertile women

Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background: Infertility is a public health problem worldwide. One of the important factors involved in infertility is the poor quality of endometrium which leads to death of the ovum before and after implantation. Glycogen is known to be a direct source of nutrients for the early conceptus and its depletion may result in inadequate preparation of endometrium around the time of implantation and leading to infertility.


INTRODUCTION
Infertility affects approximately 8%-10% of couples worldwide and India alone accounts for around 25% of cases. 1 Infertility is a complex problem requiring a battery of tests to detect the defect. Endometrial biopsy not only is a simple, economical, and valuable method of determining ovulation but also provides information about any defect in the theaters-ovarian endocrine chain. 2 One of the important factors involved in infertility is the poor quality of endometrium which leads to the death of the ovum before and after implantation. Glycogen is known to be a direct source of nutrient for the early conceptus and its depletion may result in inadequate preparation of endometrium around the time of implantation, and hence causes infertility. 3 Scarcity in the literature on the role of glycogen content in the endometrium as an etiological factor from rural south India prompted us to undertake this study to explore the role of glycogen content in the endometrium along with other factors like duration of infertility, the pattern of menstruation and other clinical features in causing infertility. 4

MATERIALS AND METHODS
The present prospective study was conducted in a tertiary care hospital which caters to a rural population in and around Hoskote, for a period of 2 years from July 2018-July 2020. Ethical clearance was obtained from the institutional ethics committee for the study. The study included 75 cases biopsied to evaluate infertility (primary and secondary). Patients who failed to conceive after one year of unprotected coitus following marriage were investigated as cases of primary infertility and patients who failed to conceive after having prior conception were investigated as cases of secondary infertility. Detailed information of the clinical history of the menstrual cycle, last menstrual period, and obstetric history were obtained.

Inclusion criteria:
Endometrial biopsies from patients with primary or secondary infertility were included.
Exclusion criteria: • Inadequate biopsies • Cases previously worked up for infertility and on treatment.
Premenstrual endometrial tissues obtained from the obstetric and gynecology department were processed and subjected to routine processing. Haematoxylin and Eosin (H and E) and Periodic Acid-Schiff (PAS) stains were performed on all the cases and analyzed. Glycogen content was graded according to Arzac and Blanchet method as given below. 5 0 = Negative reaction (no staining); + = Very small granules; ++ = Coarse granules; +++ = Small masses; ++++ = Large masses 0 and 1+ were taken as mild, ++ as moderate, +++ and ++++ were considered as high.The findings were compiled and analyzed in relation to the endometrial histology.     All the data were collected in a proforma and entered into MS Excel sheets. Descriptive statistics like mean and percentage were used for the study. The findings obtained were analyzed and compared with similar studies in the literature.

RESULTS
Amongst 75 cases of infertility, 77.3% (n=58) of cases were of primary infertility and 22.7% (n=17) of cases were of secondary infertility. The majority of patients were in the age group of 26-30 years (Table.1). The youngest patient was 21 years old and the oldest was 37 years.
Among 58 cases of primary infertility, 35 cases (60.4%) had normal cycles and among 17 cases of secondary infertility, 12 cases (70.6%) had normal cycles. In the cases with primary infertility, irregular cycles, menorrhagia, dysmenorrhea, and oligomenorrhea were the abnormalities encountered. In Secondary infertility too, irregular cycles, menorrhagia, and oligomenorrhea were noted as depicted in Table 2.
On histology, the most common finding was that of a proliferative pattern depicting anovulatory cycles. This was seen in 52% (n=39) of cases and was the most common pattern in both the primary and secondary infertility groups. A secretory pattern was observed in 38.7% of the 75 cases studied (n=29). This accounted for the next common finding in both primary and secondary infertility groups.
Luteal phase defect was seen in 4%of 75 cases studied. This accounted for 3.5% of primary and 5.8% of the secondary infertile groups. The hyperplasia group which included cases of both disordered proliferation and simple hyperplasia accounted for 4% of cases. This feature accounted for 1.7% of primary and 11.8% of secondary infertility cases. A single case of tubercular endometritis was encountered among the primary infertility cases. The findings are highlighted in Table 3 PAS staining and interpretation were done with appropriate controls for checking the adequacy of staining where in endometrium showed mild glycogen in 54.6%, moderate in 9.3%, heavy in 9.3%, and intense in 26.8% of infertility patients.

DISCUSSION
In a rural hospital setup, where immunological and hormonal assay procedures are not easily available or affordable, endometrial biopsy is an important investigation for infertility. 6 In our study, primary infertility was more common than secondary infertility i.  al6 noted a higher proportion of secondary infertility cases with luteal phase defects (16.7%). This could be due to the small sample size of secondary cases studied by them.
Tuberculous endometritis is still a major cause of infertility in developing countries and any woman with unexplained infertility should be investigated for tuberculosis. It accounted for 1.72% of our cases. Similar findings have been observed by Kafeelet al9 (0.8%) and Ahmed M etal 10 (0.51%). In contrast, Punyashetty et al 15 encountered a higher incidence of 3.9%. Variations in socioeconomic status and education could be factors responsible for the variations.
In the present study, PAS stain was done on all 75 cases. Glycogen deficiency (mild) on PAS stain is indicated by Grade 0 and +, moderate amount of glycogen as ++ and high glycogen is indicated by +++ and ++++. In our study glycogen depletion was seen in 41 cases (54.6%) whereas it was 43.3% in a study done by Girish et al 4  Genital tract glycogen is unique in that unlike muscular glycogen, it is unaffected by either carbohydrate intake or exercise. Maeyama et al 16 assessed the urinary pregnanediol and found a high correlation between the function of corpus luteum and endometrial glycogen deposition. Hughes in 1967 reported that glycogen is present in the highest concentration around the 17 to 20 the day of the cycle. 17 Interpretation of the relative finding mentioned above should be hence be done in the context of the phase of the cycle as done below, where the glycogen content is known to be low in proliferative and high in secretory phases.
All 39 cases that showed a proliferative phase on histology all had a glycogen content of (0) and (+) grade. Similar findings have been documented by Sharma et al 14 and Girish et al. 4 In the 29 cases showing secretory phase on histology a majority (89.7%) of cases showed a grade (+++) or (++++) glycogen content. A similar finding of 92.3% was noticed by Sharma et al. 14 Comparable results between the 3 studies can also be seen for luteal phase defect where all studies found reduced glycogen content of grade (+) or (++) and a slight predominance of grade (++) levels.
Unlike our study, the other studies did not report the glycogen grade for cases of tubercular endometritis or hyperplasia. In the present study, the single case of tubercular endometritis showed a secretory pattern on histology and correspondingly a glycogen grade of (+++). Endometrial hyperplasia, which is due to excess levels of estrogen can also modify the endometrial glycogen content.
In the present study, all the three cases studied had a lower glycogen content of (+) or (++). 18 have analyzed glycogen content in endometrial biopsies of infertile women in secretory phase and luteal phase defect where authors have reported a glycogen deficiency of 28.88% and 24.7% respectively. Similar findings have been noted in our study (18.7%). However, the documentation of glycopenia in 12% of all 75 cases(3 cases of secretory, 3 cases of hyperplasia, and 3 cases of luteal phase defect) studied presents the true population who would receive hormonal therapy and thereby have an improved fertility potential. In the remaining cases which showed a proliferative pattern, an additional endometrial biopsy may be required on a followup to identify the true nature of the deficiency.

CONCLUSION
Our study highlights the importance of estimating glycogen deficiency in the endometrium in cases with infertility in a rural hospital setup. Depletion of glycogen has been documented by studies in the past including the present study. As this factor can be corrected by hormonal therapy and thereby improve the fertility potential; it may be worthwhile to assess this parameter routinely in all cases of unexplained infertility. It needs to be ascertained in the future whether glycopenia is a primary feature in the biopsy or secondary to the various histological changes noted in the endometrium.