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Severe teratospermia in an infertile man with bronchiectasis
FERTILITY AND STERILITY Copyright 1985 The American Fertility Society
Vol. 44, No. 6, December 1985 Printed in U.SA.
Severe teratospermia in an infertile man with bronchiectasis
Maria Margherita de Santi, M.S. *t Concetta Gardi, M.S.t Giuseppe Lungarella, M.D.t Siena University, Siena, Italy
The association between bronchiectasis and male infertility has already been reported by several authors. 1 - 3 In particular, bronchiectasis and male infertility were commonly found in patients suffering from cystic fibrosis, 1 immotile cilia syndrome, 2 and Young's syndrome. 3 Male infertility in these patients is believed to be because of different conditions: i.e., agenesis of epididymis and vasa deferentia in cystic fibrosis, 1 immotile spermatozoa by structural defects of axoneme in immotile cilia syndrome, 2 and defective epididymal sperm transport and epididymal obstruction in Young's syndrome. 3 The present article describes the ultrastructural alterations observed in spermatozoa from an infertile man with bronchiectasis in whom the infertility is not related to the above-mentioned conditions but appears to be caused by a generalized teratospermia. The latter condition consisted of rare abnormalities heretofore undescribed in the ejaculate of an infertile man. CASE REPORT
A 34-year-old man married to a fertile woman was referred for primary infertility. Physical examination revealed no abnormalities, and the genitals appeared normal. The testes volume and serum prolactin, follicle-stimulating hormone, and luteinizing hormone values were all within normal limits. Cultures of seminal plasma failed
Received June 6, 1985; revised and accepted July 25, 1985. *Institute of Pathological Anatomy. tReprint requests: Maria Margherita de Santi, M.S., Istituto di Anatomia Patologica dell'Universita di Siena, Policlinico "le Scotte," 53100 Siena, Italy. +Institute of General Pathology. Vol. 44, No.6, December 1985
to reveal any trace of infection, and the specimens exhibited normal viscosity, liquefaction, and pH. A preliminary examination of semen sample revealed that almost all the spermatozoa had immobility and showed normal heads and several tails. Past medical history revealed several admissions to various hospitals for chonic bronchitis and pneumonia. His history was free of alcohol, tobacco consumption, and possible noxious exposures to antimicrotubule agents. In addition, he had a right lower lobectomy for bronchiectasis. Routine laboratory data, immunologic profile, and sweat chlorides were normal. Semen analysis was carried out on two different samples, and both showed a very similar pattern, as will be described later. Semen samples were collected into disposable sterile plastic containers and were allowed to liquefy at room temperature for 30 minutes. An aliquot was taken for routine semen analysis (volume, sperm count, motility, and morphologic features) according to the standard procedure. The number of flagella per cell was obtained from combined observation by light and electron microscopy (EM) according to a recently reported method. 4 The remainder of the semen samples was used for biochemical and EM analysis. For EM examination, the samples were fixed in glutaraldehyde (2.5%) buffered with 0.1 M cacodylate at pH 7.3, postfixed in Os0 4 (1 %), dehydrated in graded ethanol solutions, and embedded in Araldite (Balzers Union, Liechtenstein). Sections were cut on an LKB ultramicrotome (LKB-Produkter, Bromma, Sweden), stained with uranyl acetate and lead citrate, and examined in a Zeiss EM 109 apparatus (Zeiss OpMi, Oberkochen, FRG). Immediately after liquefaction, a part of the ejaculates was used for the electrophoretic analyde Santi et al. Communications-in-brief
849
Teratospermia was constant. About 80% of spermatozoa showed abnormal morphologic features of the heads, which appeared roundish in shape, irregularly outlined, and with a malformed acrosome (Fig. 1a). Whether or not associated with the latter malformation, a high prevalence of spermatozoa (~ 65%) presented a variable number of tails (between two and five) (Fig. 1b). The mean number of tails per head was 3.2, as obtained from the combined observation by light microscopy and EM examination. However, few cells (5%) with an immature appearance displayed a round shape and no tails. Only 5% of the cells had a normal motility, whereas most cells showed poor motility by incoordinate beating of the multiple tails. ELECTRON MICROSCOPY
Figure 1 (a), Round-headed spermatozoon with multiple tails, hemalum-eosin stain; (b), spermatozoa bearing a variable number of tails, hemalum-eosin stain.
sis of dynein high molecular weight (HMW) polypeptides of the sperm flagella. The procedure for the isolation of sperm flagella was the same described by Bell et al. 5 for preparation of sperm flagellar axonemes of Tripneustes gratilla. The axonemal pellets were suspended in saline and dissolved by boiling for 2 minutes in 0.062 M tris-HCl, pH 6.8, containing 2% sodium dodecyl sulfate, 5% mercaptoethanol, 0.01% bromophenol blue, and 10% glycerol. Electrophoresis was performed with the discontinuous high pH method of Laemli on 4% acrylamide gels. 5 Multiple nasal specimens, taken from the anterior part of the middle turbinate, were obtained by nasal biopsy at a time when the patient was clinically well. Tissue was handled and processed for light microscopy and EM. Consent for a testis biopsy was not obtained.
RESULTS LIGHT MICROSCOPY
Semen analysis revealed a slight oligospermia (49 x 10 6 /ml) and only few spermatozoa (4%) with normal morphologic features. 850
de Santi et al.
Communications-in-brief
According to the light microscopic findings, ultrastructural examination revealed a generalized teratospermia and several cells with a roundish appearance, which resembled immature spermatozoa. Teratospermia consisted of a high prevalence of spermatozoa with abnormal heads and/or with multiple tails surrounded by the same plasma membrane. Usually, in abnormal heads, the acrosome was highly developed but malformed. Often, it appeared detached from the nucleus, lying free in the cytoplasm (Fig. 2). Nuclei were abnormally outlined and sometimes showed cavities associated with fine threads of chromatin. In some cells, flattened nuclei appeared displaced from the median plane of the head because of the presence of residual cytoplasm. Abnormal composition and irregular distribution of chromatin were commonly found. Chromatin appeared incompletely condensed, punctuate, and dotted with many small and/or large vacuoles that contained membranous or granular inclusions (Fig. 2). Although in some cells with abnormal heads the neck region was lacking, in most cells this region was embedded in wide cytoplasm and filled with subcellular structures usually present in the midpiece region of the normal, mature spermatozoon. In these abnormal cells, the connecting piece was generally deranged, and the nuclear envelope frequently included several basal plates that were each associated with a connecting piece. Other cells showed several free tails that appeared to lack the basal plate, large cytoplasmic electron-dense structures resembling myelin Fertility and Sterility
ELECTROPHORETIC ANALYSIS OF DYNEIN HIGH MOLECULAR WEIGHT POLYPEPTIDES
At sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, human axonemal sperm from a control fertile man usually yields at least four major bands with various intensity in the HMW region (ranging from 300 to 400 Kd). These bands are generally believed to represent components of dynein adenosine triphosphatase molecules. No appreciable variation in intensity and migration rate for these bands was observed between axonemal samples from a control fertile man and our infertile patient. EXAMINATION OF NASAL SAMPLES
Figure 2 Tangential section through two spermatozoa with abnormal heads. In spermatozoon A, the head shows residual cytoplasm and greatly elongated acrosome. Spermatozoon B has a large, incompletely condensed nucleus containing a vacuole with membranous inclusion. Uranyl acetate-lead citrate, x 7700.
figures, and a high number of mitochondria arranged in a disorderly fashion or mitochondria grouped in clusters partially resembling the mitochondrial sheath of the midpiece region. Among these structures, there were several crosssectioned axonemes both surrounded and not surrounded by their respective outer fibers. Generally, axonemes showed loss or disorganization of peripheral microtubules; however, in a few spermatozoa, normal structured axonemes were recognized among deranged ones. The axonemes with a 9 + 2 pattern of organization usually showed a normal substructure, with well-evident dynein arms and normal radial spokes. The more apparently mature spermatozoa displayed a lessevident residual cytoplasm and single, short, and thick tails. In some cells no normal transition from midpiece to principal piece was ·observed, and the fibrous sheath extended far into the mitochondria-containing cytoplasm. In addition, free tails and isolated cytoplasmic structures filled by all kinds of tail remnants, isolated mitochondria, and isolated deranged remnants of coarse fibers or a fibrous sheath were scattered throughout. Vol. 44, No. 6, December 1985
At light microscopy, the examination of the specimens from nasal mucosa showed no remarkable changes in the respiratory epithelium. At EM examination, the only recognizable alteration was the presence of nonspecific changes in ciliary morphologic features, similar to those usually detectable in patients with recurrent respiratory disease.
DISCUSSION
Examination by EM of sperm and nasal samples in a patient suffering from infertility and bronchiectasis revealed the presence of a generalized teratospermia and no detectable specific defects in respiratory cilia. The absence in our patient of defects in the dynein structure of sperm tails and respiratory cilia as well as the absence of a high grade of oligospermia (usually characteristic of epididymal obstruction or agenesis of epididymis) strongly suggests that the association between infertility and bronchiectasis is casual and not related to a generalized syndrome like immotile cilia syndrome or Young's syndrome. Discussion on the cause of the observed abnormalities in sperm morphologic features can only be speculative. Anyway, in light of the present knowledge of spermatogenesis, the various reported abnormalities may be related to a defective function of the manchette during spermiogenesis. Such a view originates from studies in various species oflaboratory animals that involve either the selection of genetic mutants or the administration of anti-microtubule agents (for review see Escalier 4 ). The results obtained in such de Santi et al.
Communications-in-brief
851
studies offer an explanation for abnormalities comparable to the above-described alterations. In our patient, the infertility does not appear related to a single defect but to multiple defects. In fact, a high prevalence in ejaculates of roundheaded spermatozoa, changes in chromatin condensation, or tail defects (i.e., multiple tails or other abnormalities in tail structure at the level of the neck region, axoneme, and fibrous sheath) are separately reported as a cause of male infertility. Round-headed spermatozoa that lack aerosomes have been reported in the literature in no more than 30 infertile men (for review see FlorkeGerloff6), whereas a complete subcellular derangement of the ultrastructure of human spermatozoa has been reported, to our knowledge, only in one patient. 7 On the other hand, spermatozoa with multiple tails have occasionally been observed in ejaculates from infertile patients. 4 These rare abnormalities are present in most of the spermatozoa of the above-reported infertile man. SUMMARY
Morphologic examination of sperm samples from an infertile man with bronchiectasis revealed a severe teratospermia characterized by rare abnormalities that were present in most of his spermatozoa. These abnormalities were represented by round-headed spermatozoa, changes in chromatin condensation, acrosomal alterations, multiple tails, and complete subcellular derangement. The similarity between the above-re-
852
de Santi et al. Communications-in-brief
ported abnormalities- and those observed in various species of laboratory animals strongly suggests that the observed defects might be the result of a defective function of the manchette during spermiogenesis. The absence of specific defects of the axonemal morphologic features in sperm tails and in respiratory cilia, together with some laboratory data, suggests that association between infertility and bronchiectasis cannot be related, in our patient, to a generalized syndrome. REFERENCES 1. Watchti JM, Marie J: Le voies genitales males dans le maladie fibro-kystique du pancreas. Ann Pediatr (Paris) 17:490, 1970 2. Palmblad J, Mossberg B, Afzelius BA: Ultrastructural, cellular, and clinical features of the immotile-cilia syndrome. Annu Rev Med 35:481, 1984 3. Le Lannou D, Lobel B, Verger JP, Fauchet R, Genetet B, de Lebarthe B: Male infertility and chronic suppurating respiratory disease. In Progress in Reproductive Biology, Vol 8, Edited by PO Hubinont. Basel, S. Karger, 1981, p 157 4. Escalier D: Human spermatozoa with large heads and multiple flagella: a quantitative ultrastructural study of 6 cases. Bioi Cell 48:65, 1983 5. Bell GW, Fraser CL, Sale WS, Tang WJG, Gibbons IR: Preparation and purification of dynein. In Methods in Enzymology, Vol85, Edited by DW Frederiksen, LW Cunningham. New York, London, Academic Press, 1982, p 450 6. Fliirke-Gerloff S, Tiipfer-Petersen E, Muller-Esterel W, Mansouri A, Schatz R, Schirren C, Schill W, Engel W: Biochemical and genetic investigation of round-headed spermatozoa in infertile men including two brothers and their father. Andrologia 16:187, 1984 7. Pedersen H, Hammen R: Ultrastructure of human spermatozoa with complete subcellular derangement. Arch Androl 9:251, 1982
Fertility and Sterility
Vol. 44, No. 6, December 1985 Printed in U.SA.
Severe teratospermia in an infertile man with bronchiectasis
Maria Margherita de Santi, M.S. *t Concetta Gardi, M.S.t Giuseppe Lungarella, M.D.t Siena University, Siena, Italy
The association between bronchiectasis and male infertility has already been reported by several authors. 1 - 3 In particular, bronchiectasis and male infertility were commonly found in patients suffering from cystic fibrosis, 1 immotile cilia syndrome, 2 and Young's syndrome. 3 Male infertility in these patients is believed to be because of different conditions: i.e., agenesis of epididymis and vasa deferentia in cystic fibrosis, 1 immotile spermatozoa by structural defects of axoneme in immotile cilia syndrome, 2 and defective epididymal sperm transport and epididymal obstruction in Young's syndrome. 3 The present article describes the ultrastructural alterations observed in spermatozoa from an infertile man with bronchiectasis in whom the infertility is not related to the above-mentioned conditions but appears to be caused by a generalized teratospermia. The latter condition consisted of rare abnormalities heretofore undescribed in the ejaculate of an infertile man. CASE REPORT
A 34-year-old man married to a fertile woman was referred for primary infertility. Physical examination revealed no abnormalities, and the genitals appeared normal. The testes volume and serum prolactin, follicle-stimulating hormone, and luteinizing hormone values were all within normal limits. Cultures of seminal plasma failed
Received June 6, 1985; revised and accepted July 25, 1985. *Institute of Pathological Anatomy. tReprint requests: Maria Margherita de Santi, M.S., Istituto di Anatomia Patologica dell'Universita di Siena, Policlinico "le Scotte," 53100 Siena, Italy. +Institute of General Pathology. Vol. 44, No.6, December 1985
to reveal any trace of infection, and the specimens exhibited normal viscosity, liquefaction, and pH. A preliminary examination of semen sample revealed that almost all the spermatozoa had immobility and showed normal heads and several tails. Past medical history revealed several admissions to various hospitals for chonic bronchitis and pneumonia. His history was free of alcohol, tobacco consumption, and possible noxious exposures to antimicrotubule agents. In addition, he had a right lower lobectomy for bronchiectasis. Routine laboratory data, immunologic profile, and sweat chlorides were normal. Semen analysis was carried out on two different samples, and both showed a very similar pattern, as will be described later. Semen samples were collected into disposable sterile plastic containers and were allowed to liquefy at room temperature for 30 minutes. An aliquot was taken for routine semen analysis (volume, sperm count, motility, and morphologic features) according to the standard procedure. The number of flagella per cell was obtained from combined observation by light and electron microscopy (EM) according to a recently reported method. 4 The remainder of the semen samples was used for biochemical and EM analysis. For EM examination, the samples were fixed in glutaraldehyde (2.5%) buffered with 0.1 M cacodylate at pH 7.3, postfixed in Os0 4 (1 %), dehydrated in graded ethanol solutions, and embedded in Araldite (Balzers Union, Liechtenstein). Sections were cut on an LKB ultramicrotome (LKB-Produkter, Bromma, Sweden), stained with uranyl acetate and lead citrate, and examined in a Zeiss EM 109 apparatus (Zeiss OpMi, Oberkochen, FRG). Immediately after liquefaction, a part of the ejaculates was used for the electrophoretic analyde Santi et al. Communications-in-brief
849
Teratospermia was constant. About 80% of spermatozoa showed abnormal morphologic features of the heads, which appeared roundish in shape, irregularly outlined, and with a malformed acrosome (Fig. 1a). Whether or not associated with the latter malformation, a high prevalence of spermatozoa (~ 65%) presented a variable number of tails (between two and five) (Fig. 1b). The mean number of tails per head was 3.2, as obtained from the combined observation by light microscopy and EM examination. However, few cells (5%) with an immature appearance displayed a round shape and no tails. Only 5% of the cells had a normal motility, whereas most cells showed poor motility by incoordinate beating of the multiple tails. ELECTRON MICROSCOPY
Figure 1 (a), Round-headed spermatozoon with multiple tails, hemalum-eosin stain; (b), spermatozoa bearing a variable number of tails, hemalum-eosin stain.
sis of dynein high molecular weight (HMW) polypeptides of the sperm flagella. The procedure for the isolation of sperm flagella was the same described by Bell et al. 5 for preparation of sperm flagellar axonemes of Tripneustes gratilla. The axonemal pellets were suspended in saline and dissolved by boiling for 2 minutes in 0.062 M tris-HCl, pH 6.8, containing 2% sodium dodecyl sulfate, 5% mercaptoethanol, 0.01% bromophenol blue, and 10% glycerol. Electrophoresis was performed with the discontinuous high pH method of Laemli on 4% acrylamide gels. 5 Multiple nasal specimens, taken from the anterior part of the middle turbinate, were obtained by nasal biopsy at a time when the patient was clinically well. Tissue was handled and processed for light microscopy and EM. Consent for a testis biopsy was not obtained.
RESULTS LIGHT MICROSCOPY
Semen analysis revealed a slight oligospermia (49 x 10 6 /ml) and only few spermatozoa (4%) with normal morphologic features. 850
de Santi et al.
Communications-in-brief
According to the light microscopic findings, ultrastructural examination revealed a generalized teratospermia and several cells with a roundish appearance, which resembled immature spermatozoa. Teratospermia consisted of a high prevalence of spermatozoa with abnormal heads and/or with multiple tails surrounded by the same plasma membrane. Usually, in abnormal heads, the acrosome was highly developed but malformed. Often, it appeared detached from the nucleus, lying free in the cytoplasm (Fig. 2). Nuclei were abnormally outlined and sometimes showed cavities associated with fine threads of chromatin. In some cells, flattened nuclei appeared displaced from the median plane of the head because of the presence of residual cytoplasm. Abnormal composition and irregular distribution of chromatin were commonly found. Chromatin appeared incompletely condensed, punctuate, and dotted with many small and/or large vacuoles that contained membranous or granular inclusions (Fig. 2). Although in some cells with abnormal heads the neck region was lacking, in most cells this region was embedded in wide cytoplasm and filled with subcellular structures usually present in the midpiece region of the normal, mature spermatozoon. In these abnormal cells, the connecting piece was generally deranged, and the nuclear envelope frequently included several basal plates that were each associated with a connecting piece. Other cells showed several free tails that appeared to lack the basal plate, large cytoplasmic electron-dense structures resembling myelin Fertility and Sterility
ELECTROPHORETIC ANALYSIS OF DYNEIN HIGH MOLECULAR WEIGHT POLYPEPTIDES
At sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, human axonemal sperm from a control fertile man usually yields at least four major bands with various intensity in the HMW region (ranging from 300 to 400 Kd). These bands are generally believed to represent components of dynein adenosine triphosphatase molecules. No appreciable variation in intensity and migration rate for these bands was observed between axonemal samples from a control fertile man and our infertile patient. EXAMINATION OF NASAL SAMPLES
Figure 2 Tangential section through two spermatozoa with abnormal heads. In spermatozoon A, the head shows residual cytoplasm and greatly elongated acrosome. Spermatozoon B has a large, incompletely condensed nucleus containing a vacuole with membranous inclusion. Uranyl acetate-lead citrate, x 7700.
figures, and a high number of mitochondria arranged in a disorderly fashion or mitochondria grouped in clusters partially resembling the mitochondrial sheath of the midpiece region. Among these structures, there were several crosssectioned axonemes both surrounded and not surrounded by their respective outer fibers. Generally, axonemes showed loss or disorganization of peripheral microtubules; however, in a few spermatozoa, normal structured axonemes were recognized among deranged ones. The axonemes with a 9 + 2 pattern of organization usually showed a normal substructure, with well-evident dynein arms and normal radial spokes. The more apparently mature spermatozoa displayed a lessevident residual cytoplasm and single, short, and thick tails. In some cells no normal transition from midpiece to principal piece was ·observed, and the fibrous sheath extended far into the mitochondria-containing cytoplasm. In addition, free tails and isolated cytoplasmic structures filled by all kinds of tail remnants, isolated mitochondria, and isolated deranged remnants of coarse fibers or a fibrous sheath were scattered throughout. Vol. 44, No. 6, December 1985
At light microscopy, the examination of the specimens from nasal mucosa showed no remarkable changes in the respiratory epithelium. At EM examination, the only recognizable alteration was the presence of nonspecific changes in ciliary morphologic features, similar to those usually detectable in patients with recurrent respiratory disease.
DISCUSSION
Examination by EM of sperm and nasal samples in a patient suffering from infertility and bronchiectasis revealed the presence of a generalized teratospermia and no detectable specific defects in respiratory cilia. The absence in our patient of defects in the dynein structure of sperm tails and respiratory cilia as well as the absence of a high grade of oligospermia (usually characteristic of epididymal obstruction or agenesis of epididymis) strongly suggests that the association between infertility and bronchiectasis is casual and not related to a generalized syndrome like immotile cilia syndrome or Young's syndrome. Discussion on the cause of the observed abnormalities in sperm morphologic features can only be speculative. Anyway, in light of the present knowledge of spermatogenesis, the various reported abnormalities may be related to a defective function of the manchette during spermiogenesis. Such a view originates from studies in various species oflaboratory animals that involve either the selection of genetic mutants or the administration of anti-microtubule agents (for review see Escalier 4 ). The results obtained in such de Santi et al.
Communications-in-brief
851
studies offer an explanation for abnormalities comparable to the above-described alterations. In our patient, the infertility does not appear related to a single defect but to multiple defects. In fact, a high prevalence in ejaculates of roundheaded spermatozoa, changes in chromatin condensation, or tail defects (i.e., multiple tails or other abnormalities in tail structure at the level of the neck region, axoneme, and fibrous sheath) are separately reported as a cause of male infertility. Round-headed spermatozoa that lack aerosomes have been reported in the literature in no more than 30 infertile men (for review see FlorkeGerloff6), whereas a complete subcellular derangement of the ultrastructure of human spermatozoa has been reported, to our knowledge, only in one patient. 7 On the other hand, spermatozoa with multiple tails have occasionally been observed in ejaculates from infertile patients. 4 These rare abnormalities are present in most of the spermatozoa of the above-reported infertile man. SUMMARY
Morphologic examination of sperm samples from an infertile man with bronchiectasis revealed a severe teratospermia characterized by rare abnormalities that were present in most of his spermatozoa. These abnormalities were represented by round-headed spermatozoa, changes in chromatin condensation, acrosomal alterations, multiple tails, and complete subcellular derangement. The similarity between the above-re-
852
de Santi et al. Communications-in-brief
ported abnormalities- and those observed in various species of laboratory animals strongly suggests that the observed defects might be the result of a defective function of the manchette during spermiogenesis. The absence of specific defects of the axonemal morphologic features in sperm tails and in respiratory cilia, together with some laboratory data, suggests that association between infertility and bronchiectasis cannot be related, in our patient, to a generalized syndrome. REFERENCES 1. Watchti JM, Marie J: Le voies genitales males dans le maladie fibro-kystique du pancreas. Ann Pediatr (Paris) 17:490, 1970 2. Palmblad J, Mossberg B, Afzelius BA: Ultrastructural, cellular, and clinical features of the immotile-cilia syndrome. Annu Rev Med 35:481, 1984 3. Le Lannou D, Lobel B, Verger JP, Fauchet R, Genetet B, de Lebarthe B: Male infertility and chronic suppurating respiratory disease. In Progress in Reproductive Biology, Vol 8, Edited by PO Hubinont. Basel, S. Karger, 1981, p 157 4. Escalier D: Human spermatozoa with large heads and multiple flagella: a quantitative ultrastructural study of 6 cases. Bioi Cell 48:65, 1983 5. Bell GW, Fraser CL, Sale WS, Tang WJG, Gibbons IR: Preparation and purification of dynein. In Methods in Enzymology, Vol85, Edited by DW Frederiksen, LW Cunningham. New York, London, Academic Press, 1982, p 450 6. Fliirke-Gerloff S, Tiipfer-Petersen E, Muller-Esterel W, Mansouri A, Schatz R, Schirren C, Schill W, Engel W: Biochemical and genetic investigation of round-headed spermatozoa in infertile men including two brothers and their father. Andrologia 16:187, 1984 7. Pedersen H, Hammen R: Ultrastructure of human spermatozoa with complete subcellular derangement. Arch Androl 9:251, 1982
Fertility and Sterility