Maturation of Reproductive Functions in Chinese Adolescents
A group of Chinese school boys aged 10-16 years old were investigated for their emission of spermatozoa in urine "spermaturia" and the urinary excretion of testosterone (T) levels. Nuclear areas and relative DNA contents of spermatozoa in urine were also measured. Results showed that the median age of onset of spermaturia was 13.7 years. Frequency of spermaturia increased with ages between 11-16 years; it also increased with pubertal stages between Tanner stages 3 to 5 (G3-G5). 67% of the boys studied showed spermaturia in 6 consecutive days of their urine samples; 10% of those in pubertal stages G4 and G5 showed spermaturia 3 to 5 times in 6 days. Levels of urinary T from positive samples were higher than that from the negative ones. Also urinary T was higher in patients in whom spermaturia revealed more than once during the study period. Another group of the teenage school girls (10-15 years old) in our longitudinal survey showed that the menarcheal age was 12.51±0.97 years. Ovulatory cycles of post menarcheal girls were studied by serial urinary progesterone (P) levels determined between two menstrual cycles and covering both the luteal (L) and the follicular (F) phases. Urinary P levels were corrected by creatinine (Cr) concentration. By using the ratio of PL/Cr to PF/Cr > 2 as a criteria for identification of ovulatory cycles according to the data in adult women controls, we found the rates of ovulatory cycles to be 8.1, 11.9, 35.7 and 46.2 % for girls with gynecologic age of 0.5, 1.0, 2.0 and 2.5 years respectively. Ovulation might start as early as gynecologic age of 0.04-0.3 years during the 2nd or the 4th post-menarcheal cycle. We concluded that the non invasive method utilizing multiple urine samples, for assays of spermaturia and hormonal (T,P) measurements could be effectively used in the study of maturation of adolescent reproductive functions.
Keyword : Adolescent; Ovulatory cycle; Spermaturia
The onset of puberty is a critical period of physical, biological and psychosocial changes associated with adolescents. Factors such as apparent trend in the lowering of the age of menarche,1 exposure to western culture and life style, changes in values brought about by increasing urbanization operating in developing societies contributed to reproductive related health risks for adolescents. Sexual promiscuity among teenagers was nowadays universally believed to be increasing with considerable hazards of illegitimate pregnancy, illegal abortion and delinquency in adolescence.2 Thus effort in promoting reproductive health and sex education in China to-day seems more important than ever before. What is the process of sexual maturation in Chinese teenagers? It is essential for teachers, parents and social workers or youth health to have a comprehensive understanding.
In this study, by means of urinary samples, we could be able to detect the spermatozoa emission in urine "spermaturia" and urinary testosterone and progesterone levels for the evaluation of onset of spermatogenesis and ovulation during puberty stages.
Subjects in Cohorts
For spermaturia studies
For menstrual cycles and ovulation studies
Emission of spermatozoa in urine was detected using light microscope by examining the first portion of the overnight morning voided urine collected over a 6 consecutive days period as a batch. Urine was freshly prepared, centrifuged and examined under standard condition. Part of one of the batch urinary samples was denoted to testosterone (T) determination by radio-immuno assay (RIA)3 and creatinine (Cr) content by automatic biochemistry analysor. Ratio of urinary T to Cr content was calculated. Nuclear surface areas and DNA relative contents of sperms in urine were measured by pretreatment with Feulgen stain on the drops of the urinary sediment on the slide. Records and analysis were performed by microspectrophotometer scan through digitalizing image analysis by VIDAS system (OPTON west Germany).
Teenage menarcheal ages and menstrual cycle patterns were investigated through longitudinal survey every 6 months and menstrual diaries records checked every 3 months. Ovulatory cycles were studied by serial urinary progesterone (P) determinations by RIA4 from consecutive weekly collected overnight morning voided urine between interval of at least two menses periods so as to cover both the follicular phase (F) and luteal phase (L) of the cycle. Urinary P level was corrected by ratio with creatinine content in the same sample. Criterion for identification of ovulatory cycle was based upon the ratio of urinary PL/Cr to PF/Cr > 2 which was verified in normal control women while their plasma P level in luteal phase of the ovulatory cycle being >3mg%.
By the cross sectional study of spermaturia in 575 boys, positive results were revealed in 189 boys (32.9%) and in 36 control adults (62%). Rates of positive findings were 10%, 37% and 55% according to age groups from 11-, 13- and 15- years old, 11%, 37% and 50% according to puberty stages from G3-G5 (Tanner) respectively. Difference was significant among percentages of spermaturia revealed by various age, groups and puberty stages (Fig. 1, 2, p < 0.001 by X2 test). Frequency of positive spermaturia within batch samples was mostly once (67%) but 3-5 times in 10% of boys in puberty stage G4 or G5 (Fig. 3). In adult controls positive findings were in 65% as once within 6 days batch samples examined but non of them showing frequency more than twice.
By the longitudinal studies of 164 boys, median age of spermaturia started at 13.7 years during puberty stage G3-G4. Urinary T levels significantly increased according ages (Table I) and revealed significant difference between values for positive and negative samples for spermaturia (Table II). Urinary T levels were also found with significant difference between values as nmol/L for higher positive frequency (3-4 times within a batch) and just once, but not shown by values in pmol/mg Cr (Table III).
Mean spermatozoa nuclear DNA relative contents and surface areas for boys aged 11-16 years and control adults were shown in Table IV. Comparison of values of each age group with that of the adults all showed no significant difference.
Mean menarcheal age was 12.51±0.97 years through longitudinal survey of 144 teenage girls. Using criterion as urinary PL/Cr with ratio to PF/Cr >2 as ovulatory cycles, we identified 90% of the menstrual cycles being ovulatory for normal control women and 8.1%, 11.9%, 35.7% and 46.2% for the teenage girls during their gynecologic age 0.5, 1.0, 2.0 and 2.5 years respectively (Fig. 4). Individual girls started ovulation even earlier during the 2nd or the 4th post menarcheal cycles or with gynecologic ages just 0.04 and 0.3 years. More than half (55%) of the ovulatory menstrual cycles were with regular intervals of 26-35 days between menses. Shorter intervals show remarkable declined rate of ovulation (Figure 5) . Regular menses were also found coexisted with costant ovulation in 78.1% of the menstrual cycles.
Onset of spermatozoa emission "spermarche" was suspected as valid criterion or hall mark for sufficient sex maturity in male reproductive system.5-6 Ejaculation by masturbation in adolescent boys aiming to obtain semen samples for analysis was impracticable. It was neither reliable by retrospective questionnaires for the first ejaculation because of uncertainty of existence of spermatozoa.
Emission of spermatozoa in urine "spermaturia" was the primary finding by Baldwin5 and was adapted as non offensive method in adolescent andrology study. The mechanism was presumed as consequence of urethral washing of ejaculum remnants. The truth that spermaturia validated only when positive result obtained, as such result could not be false for indicating spermatogenesis. On contrary negative result could not be surely regarded as lack of spermatozoa. The false negative result might be due to post masturbation or nocturnal emission urine voided prior to the first morning void or later void urine with spermatozoa already rinsed out in the void immediately following emission. Insufficient amount of spermatozoa produced or less frequency of urine samplings also reduced chance to be positive.7-9
Our findings on the onset of spermaturia in school boys with references to their ages, puberty stages and levels of urinary T excretion corresponded to histologic descriptions by Sniffen,7 Charny et al10 who showed increment of seminiferons tubular diameters and maturation of spermatogenesis in proceeding from age 12-16 years old. Few of them started early quite from 11 years old as in ours.
The High frequency of positive spermaturia within the 6 days' observation in 10% of the teenagers boys in our group, lead to suspiciar that sexual behavior were quite active in the adolescent period and adequate sex and moral education induced in due time would be needed.
Are the spermatozoa in adolescent boys fertile? It remains uncertain. The fact that few young men fathered a child while they were teenagers and there is an apparent lag period between spermatozoa production and fatherhood, a male adolescent sterile period was strongly suspected.11 Many factors may be involved for its explanation. In this study although we detected no significant difference between adolescents and adults in their urinary spermatozoa nuclear DNA relative contents and surface areas by image digitalized analysis, we have noticed a wide variation in sperm head shapes and sizes, pre-dominantly in the young age group by light microscopic examination (not shown). We have not observed these for spermatozoa mobility in teenage boys which had been suspected in major role in fertilization in adults.12
While various physiological studies about the sex steroids and ovary functions were bumper in women, few numerous studies were found in the adolescent girls in particular the status of ovulation in post menarcheal menstrual cycles. Also, rates of ovulatory cycles in post menarcheal years differed widely by various authors.13-14 This might be due to difference in criterion used and sampling methodology. To assure ovulatory cycles in adult women serial measure of serum P levels within cycles was one of the convential methods applicated. The ratio of P level in mid-luteal phase (PL) to follicular phase (PF) up to 1.5-2 or only a single PL value > 3 ng/ml had been adapted to define ovulatory cycles in adults.15-16 Apter et al had suggested serum PL > 2 ng/ml as adolescent ovulation criterion.13 We propose the tentative criterion for ovulation in adolescents as ratio of urinary PL/Cr to PF/Cr> 2 which referred to the serum PL level > 3 ng/ml in our control women with regular menses and enrolled in combination study with their urinary samples in the mean time. The reliability and practicability of determing P levels in urine instead of blood had been well demonstrated, not only the serum P concentration intimately correlated with P excretion in urine, but also same pattern of P variation in urine as in blood with the two phases of menstrual cycles.17 The ovulatory cycles rate of teenagers in our group show of similar results according to their gynecologic ages with that of Apter's studies,13,18 i.e. 11.9% versus 14%, for the first gynecologic year and 46.2% versus 45% for the 2.5 and three years of menarcheal life respectively.
From this study we concluded that the non-invasive methods with repeated urinary sampleing for assays of spermaturia and hormonal (T, P) measurements were practicable and effectively applicated in the study of maturation of male and female adolescent reproductive functions.
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